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Sample records for pha-specific acetoacetyl coenzyme

  1. Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway.

    PubMed

    Okamura, Eiji; Tomita, Takeo; Sawa, Ryuichi; Nishiyama, Makoto; Kuzuyama, Tomohisa

    2010-06-22

    Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-beta-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms. The de novo synthesis of acetoacetyl-CoA is usually catalyzed by acetoacetyl-CoA thiolase via a thioester-dependent Claisen condensation reaction between two molecules of acetyl-CoA. Here, we report that nphT7, found in the mevalonate pathway gene cluster from a soil-isolated Streptomyces sp. strain, encodes an unusual acetoacetyl-CoA synthesizing enzyme. The recombinant enzyme overexpressed in Escherichia coli catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) resulted in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity was detected through the condensation of two molecules of acetyl-CoA. Based on these properties of NphT7, we propose to name this unusual enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Coexpression of nphT7 with the HMG-CoA synthase gene and the HMG-CoA reductase gene in a heterologous host allowed 3.5-fold higher production of mevalonate than when only the HMG-CoA synthase and HMG-CoA reductase genes were expressed. This result suggests that nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-beta-hydroxybutyrate. PMID:20534558

  2. Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

    SciTech Connect

    Cary, J.W.; Petersen, D.J.; Bennett, G.N. ); Papoutsakis, E.T. )

    1990-06-01

    Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.

  3. Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

    PubMed Central

    Fukao, T; Yamaguchi, S; Wakazono, A; Orii, T; Hoganson, G; Hashimoto, T

    1994-01-01

    We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation. Images PMID:7907600

  4. Site-specific cleavage of acetoacetyl-CoA synthetase by legumain.

    PubMed

    Hasegawa, Shinya; Inoue, Daiki; Yamasaki, Masahiro; Li, Chuan; Imai, Masahiko; Takahashi, Noriko; Fukui, Tetsuya

    2016-06-01

    Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme and is responsible for the synthesis of cholesterol and fatty acids. We have previously shown that AACS is cleaved by legumain, a lysosomal asparaginyl endopeptidase. In this study, we attempted to determine the cleavage site of AACS. Mutagenesis analysis of AACS revealed that Asn547 is the specific cleavage site of AACS in mouse livers. The cleaved form of AACS (1-547) lost the ability to convert acetoacetate to acetoacetyl-CoA. Moreover, hydrodynamics-based gene transduction showed that overexpression of AACS (1-547) increases the protein expression of caveolin-1, the principal component of the caveolae. These results suggest that cleavage of AACS by legumain is critical for the regulation of enzymatic activity and results in gain-of-function changes. PMID:27129883

  5. Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol.

    PubMed

    Isezaki, Nana; Sekiba, Atsushi; Itagaki, Shoko; Nagayama, Koki; Ochiai, Hiroshi; Ohmachi, Tetsuo

    2015-07-01

    Acetoacetyl-CoA thiolase is an enzyme that catalyses both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium discoideum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using a fusion protein with GFP, and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution to the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondrial targeting signal (MTS). These results indicate that DdAcat is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol using both PTS-2 and MTS signals, which overlap each other near the N-terminus, and the alternative utilization of start codons. PMID:25911059

  6. Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

    PubMed Central

    Dyer, James H.; Maina, Anthony; Gomez, Iris D.; Cadet, Melissa; Oeljeklaus, Silke; Schiedel, Anke C.

    2009-01-01

    Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT. PMID:20011134

  7. Coenzyme Q10 Therapy

    PubMed Central

    Garrido-Maraver, Juan; Cordero, Mario D.; Oropesa-Ávila, Manuel; Fernández Vega, Alejandro; de la Mata, Mario; Delgado Pavón, Ana; de Miguel, Manuel; Pérez Calero, Carmen; Villanueva Paz, Marina; Cotán, David; Sánchez-Alcázar, José A.

    2014-01-01

    For a number of years, coenzyme Q10 (CoQ10) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in blood plasma, and extensively investigated its antioxidant role. These 2 functions constitute the basis for supporting the clinical use of CoQ10. Also, at the inner mitochondrial membrane level, CoQ10 is recognized as an obligatory cofactor for the function of uncoupling proteins and a modulator of the mitochondrial transition pore. Furthermore, recent data indicate that CoQ10 affects the expression of genes involved in human cell signaling, metabolism and transport, and some of the effects of CoQ10 supplementation may be due to this property. CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, aging-related oxidative stress and carcinogenesis processes, and also statin treatment. Many neurodegenerative disorders, diabetes, cancer, and muscular and cardiovascular diseases have been associated with low CoQ10 levels as well as different ataxias and encephalomyopathies. CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral administration of CoQ10 is a frequent antioxidant strategy in many diseases that may provide a significant symptomatic benefit. PMID:25126052

  8. Membrane reactors for continuous coenzyme regeneration

    NASA Astrophysics Data System (ADS)

    Wandrey, C.; Wichmann, R.

    1982-12-01

    The importance of continuous coenzyme regeneration is discussed with respect to chemical reaction engineering. The benefit of coenzymes covalently bound to water soluble polymers is especially stressed. The performance of membrane reactors for coenzyme regeneration is discussed in comparison with other reactor concepts. The coenzyme dependent production of L-amino acids from the corresponding alpha-keto acids is used to illustrate how precise turnover numbers as a function of enzyme/coenzyme ratio, initial substrate concentration, and conversion are obtained. Thus, it becomes possible to develop a concept for optimal operating points with respect to enzyme, coenzyme, and substrate costs per unit weight of product.

  9. Characterization of cyclo-Acetoacetyl-L-Tryptophan Dimethylallyltransferase in Cyclopiazonic Acid Biosynthesis: Substrate Promiscuity and Site Directed Mutagenesis Studies

    PubMed Central

    Liu, Xinyu; Walsh, Christopher T.

    2009-01-01

    The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase with a unique pentacyclic indole tetramic acid scaffold is assembled by a three enzyme pathway CpaS, CpaD and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form β-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with eleven homologous fungal Trp-DMATs revealed five regions of conservation suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity. This motif characerization will enable a gene probe-based approach to discover additional biosynthetic Trp-DMATs. PMID:19877600

  10. Coenzyme Q and Mitochondrial Disease

    ERIC Educational Resources Information Center

    Quinzii, Catarina M.; Hirano, Michio

    2010-01-01

    Coenzyme Q[subscript 10] (CoQ[subscript 10]) is an essential electron carrier in the mitochondrial respiratory chain and an important antioxidant. Deficiency of CoQ[subscript 10] is a clinically and molecularly heterogeneous syndrome, which, to date, has been found to be autosomal recessive in inheritance and generally responsive to CoQ[subscript…

  11. Acetoacetylation of insulin

    PubMed Central

    Lindsay, D. G.; Shall, S.

    1969-01-01

    Insulin was treated with diketen at pH6·9. The reaction mixture was resolved into four components by DEAE-Sephadex chromatography. The first component was unchanged insulin. The second and third components were shown by end-group analysis to be substituted on phenylalanine B-1 and glycine A-1 respectively. The fourth component was disubstituted on both phenylalanine B-1 and glycine A-1. The ∈-amino group of lysine B-29 was not involved in the reaction at low reagent concentrations. The purity of these derivatives was checked by their electrophoretic behaviour and by measurement of the rate of their reaction with trinitrobenzenesulphonic acid. The hormonal activity of the derivatives was determined. The effect of the modifications on the hormonal activity and the tertiary structure of insulin is discussed. ImagesFig. 10. PMID:5353531

  12. Coenzyme q 10 : a review.

    PubMed

    Singh, Deependra; Jain, Vandana; Saraf, Swarnlata; Saraf, S

    2002-10-01

    Ubiquinone or Co Q(10) is essentially a vitamin like substance and is a cofactor of an enzyme. It is an integral part of the memberanes of mitocondria where it is involved in the energy production. It is a nutrient necessary for the function of every cell of the body especially vital organs of the body like heart, liver, brain etc. Studies have shown that coenzyme Q(10) alters the natural history of cardiovascular illness and has the potential of prevention of cardiovascular diseases through the inhibition of LDL cholesterol oxidation by maintenance of optimal cellular and mitochondrial function throughout the ravages of time internal and external stress. PMID:22557086

  13. Kinetics and catalytic properties of coenzyme A transferase from Peptostreptococcus elsdenii.

    PubMed Central

    Schulman, M; Valentino, D

    1976-01-01

    Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii was purified to homogeneity, and some of its physical and catalytic properties were determined. The native enzyme has a molecular weight of 181,000 and is composed of two alpha subunits (molecular weight, 65,000) and one beta subunit (molecular weight 50,000). Heat treatment of the crude cell extract to 58 degrees C causes proteolysis of the native enzyme and yields a catalytically active enzyme with an approximate molecular weight of 120,000. The native CoA transferase is specific for CoA esters of short-chain alkyl monocarboxylic acids. With acetate as CoA acceptor the enzyme is active with propionyl-, butyryl-, isobutyryl-, valeryl-, isovaleryl,- and hexanoyl-CoA but not with heptanoyl or longer-chain CoA esters. There is no activity with acetoacetyl-CoA or the CoA esters of dicarboxylic acids. Steady-state kinetics indicated that the reaction proceeds via a classical bi-, bi-ping-pong mechanism. Maximal activity is obtained with propionyl- or butyryl-CoA, and both the Vmax and Km decrease as the alkyl chain length of the CoA ester increases. All CoA esters apompetitive inhibitor although it is not active as a substrate. Evidence for an enzyme CoA intermediate was provided by demonstration of an exchange between 14C-free acids (acetate and butyrate) and their corresponding CoA esters and by isolation of a 3H-labeled CoA enzyme after incubation of the enzyme with 3H-labeled acetyl-CoA. Approximately 2 mol of CoA was bound per mol of enzyme. Images PMID:977540

  14. Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase, an enzyme of isopentenyl diphosphate biosynthesis.

    PubMed

    Sutherlin, Autumn; Hedl, Matija; Sanchez-Neri, Barbara; Burgner, John W; Stauffacher, Cynthia V; Rodwell, Victor W

    2002-08-01

    Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis. PMID:12107122

  15. Self-Incorporation of Coenzymes by Ribozymes

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNAcatalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

  16. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    PubMed

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. PMID:27372278

  17. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development

    SciTech Connect

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J.

    2012-03-31

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT‐encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T‐DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol‐localized, mevalonate‐derived isoprenoid biosynthetic pathway.

  18. Crystal structure of enoyl-coenzyme A (CoA) hydratase at 2.5 angstroms resolution: a spiral fold defines the CoA-binding pocket.

    PubMed Central

    Engel, C K; Mathieu, M; Zeelen, J P; Hiltunen, J K; Wierenga, R K

    1996-01-01

    The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA) hydratase complexed with the potent inhibitor acetoacetyl-CoA has been refined at 2.5 angstroms resolution. This enzyme catalyses the reversible addition of water to alpha,beta-unsaturated enoyl-CoA thioesters, with nearly diffusion-controlled reaction rates for the best substrates. Enoyl-CoA hydratase is a hexamer of six identical subunits of 161 kDa molecular mass for the complex. The hexamer is a dimer of trimers. The monomer is folded into a right-handed spiral of four turns, followed by two small domains which are involved in trimerization. Each turn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereochemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry. It confirms the importance of Glu164 as the catalytic acid for providing the alpha-proton during the hydratase reaction. It also shows the importance of Glu144 as the catalytic base for the activation of a water molecule in the hydratase reaction. The comparison of an unliganded and a liganded active site within the same crystal form shows a water molecule in the unliganded subunit. This water molecule is bound between the two catalytic glutamates and could serve as the activated water during catalysis. Images PMID:8895557

  19. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  20. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  1. Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

    PubMed Central

    Weichler, Maria-Teresa; Kurteva-Yaneva, Nadya; Przybylski, Denise; Schuster, Judith; Müller, Roland H.; Harms, Hauke

    2015-01-01

    The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502–15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of

  2. [Coenzyme metabolic therapy in infectious allergic myocarditis].

    PubMed

    Mazurets, A F; Gurevich, M A; Kubyshkin, V F; Dziuba, M V; Vikharev, N P

    1995-01-01

    A trial was performed of clinical efficacy of the coenzyme complex incorporating piridoxalphosphate, cobamamide and phosphaden in patients with infectious allergic myocarditis. Myo- cardial dystrophy and correlations of the myocardial enzymatic status with blood lymphocytes in the above patients were taken in consideration. Corrective action of metabolic therapy on myocardial bioenergy was coupled with positive antiarrhythmic and cardiotonic effects. Cytochemical follow-up investigations enabled long-term monitoring over the patients' condition and further catamnesis. PMID:8815275

  3. Better than Nature: Nicotinamide Biomimetics That Outperform Natural Coenzymes.

    PubMed

    Knaus, Tanja; Paul, Caroline E; Levy, Colin W; de Vries, Simon; Mutti, Francesco G; Hollmann, Frank; Scrutton, Nigel S

    2016-01-27

    The search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. Redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. Stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. Here, we investigate the efficiency of man-made synthetic biomimetics of the natural coenzymes NAD(P)H in redox biocatalysis. Extensive studies with a range of oxidoreductases belonging to the "ene" reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase XenA with selected biomimetics. In selected cases, these biomimetics outperform the natural coenzymes. "Better-than-Nature" biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost. PMID:26727612

  4. Prebiotic syntheses of vitamin coenzymes: I. Cysteamine and 2-mercaptoethanesulfonic acid (coenzyme M)

    NASA Technical Reports Server (NTRS)

    Miller, S. L.; Schlesinger, G.

    1993-01-01

    The reaction of NH3 and SO3(2-) with ethylene sulfide is shown to be a prebiotic synthesis of cysteamine and 2-mercaptoethanesulfonic acid (coenzyme M). A similar reaction with ethylene imine would give cysteamine and taurine. Ethylene oxide would react with NH3 and N(CH3)3 to give the phospholipid components ethanolamine and choline. The prebiotic sources of ethylene sulfide, ethylene imine and ethylene oxide are discussed. Cysteamine itself is not a suitable thioester for metabolic processes because of acyl transfer to the amino group, but this can be prevented by using an amide of cysteamine. The use of cysteamine in coenzyme A may have been due to its prebiotic abundance. The facile prebiotic synthesis of both cysteamine and coenzyme M suggests that they were involved in very early metabolic pathways.

  5. Elucidation of methanogenic coenzyme biosyntheses: from spectroscopy to genomics.

    PubMed

    Graham, David E; White, Robert H

    2002-04-01

    Methanogenesis, the anaerobic production of methane from CO2 or simple carbon compounds, requires seven organic coenzymes. This review describes pathways for the biosynthesis of methanofuran, 5,6,7,8-tetrahydromethanopterin, coenzyme F420, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoyl-L-threonine phosphate). Spectroscopic evidence for the pathways is reviewed and recent efforts are described to identify and characterize the biosynthetic enzymes from methanogenic archaea. The literature from 1971 to September 2001 is reviewed, and 169 references are cited. PMID:12013276

  6. Prebiotic syntheses of vitamin coenzymes: II. Pantoic acid, pantothenic acid, and the composition of coenzyme A

    NASA Technical Reports Server (NTRS)

    Miller, S. L.; Schlesinger, G.

    1993-01-01

    Pantoic acid can by synthesized in good prebiotic yield from isobutyraldehyde or alpha-ketoisovaleric acid + H2CO + HCN. Isobutyraldehyde is the Strecker precursor to valine and alpha-ketoisovaleric acid is the valine transamination product. Mg2+ and Ca2+ as well as several transition metals are catalysts for the alpha-ketoisovaleric acid reaction. Pantothenic acid is produced from pantoyl lactone (easily formed from pantoic acid) and the relatively high concentrations of beta-alanine that would be formed on drying prebiotic amino acid mixtures. There is no selectivity for this reaction over glycine, alanine, or gamma-amino butyric acid. The components of coenzyme A are discussed in terms of ease of prebiotic formation and stability and are shown to be plausible choices, but many other compounds are possible. The gamma-OH of pantoic acid needs to be capped to prevent decomposition of pantothenic acid. These results suggest that coenzyme A function was important in the earliest metabolic pathways and that the coenzyme A precursor contained most of the components of the present coenzyme.

  7. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C. Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease. PMID:19141679

  8. Electronic Structure of B12 coenzymes

    NASA Astrophysics Data System (ADS)

    Ouyang, Lizhi; Ching, W. Y.; Randaccio, Lucio

    2001-06-01

    We have carried out an ab-initio local density functional calculations of the two most important B12 coenzymes, adoensyl-cobalamin (Ado-Cbl) and methyl-cobalamin (Me-Cbl). The crystal structures were determined by accurate X-ray synchrotron radiation measurements. Both crystals have space group P2121 with four molecules, or about 800 atoms, per unit cell. Our electronic structure calculation is based on one full molecule including the side chains. Results are analyzed in terms of atom and orbital resolved partial density of states (PDOS), Mulliken effective charges and bond orders. The PDOS analysis shows that the Co complexes of both B12 coenzymes had a HOMO/LUMO gap of about 1.5 eV. The Co-C bond order in Me-Cbl is smaller than that in Ado-Cbl. This appears to be in contradiction with the measured bond dissociated energies. However, this could also indicate the importance of the effects of solvents, which were not included in the calculation. We are investigating whether the effect of the solvents could dramatically modify the electronic structures of Ado-Cbl and Me-Cbl.

  9. [Hplc estimation of coenzyme Q(10) redox status in plasma after intravenous coenzyme Q(10) administration].

    PubMed

    Kalenikova, E I; Kharitonova, E V; Gorodetskaya, E A; Tokareva, O G; Medvedev, O S

    2015-01-01

    The pharmacokinetics of the total pool of coenzyme Q(10) (Co(10)), its oxidized (ubiquinone) and reduced (ubiquinol, CoQ(10)H₂) forms have been investigated in rats plasma during 48 h after a single intravenous injection of a solution of solubilized CoQ(10) (10 mg/kg) to rats. Plasma levels of CoQ(10) were determined by HPLC with spectrophotometric and coulometric detection. In plasma samples taken during the first minutes after the CoQ(10) intravenous injection, the total pool of coenzyme Q(10) and proportion of CoQ(10)H₂ remained unchanged during two weeks of storage at -20°C. The kinetic curve of the total pool of coenzyme Q(10) corresponds to a one-part model (R² = 0.9932), while the corresponding curve of its oxidized form fits to the two-part model. During the first minutes after the injection a significant portion of plasma ubiquinone undergoes reduction, and after 7 h the concentration of ubiquinol predominates. The decrease in the total plasma coenzyme Q(10) content was accompanied by the gradual increase in plasma ubiquinol, which represented about 90% of total plasma CoQ(10) by the end of the first day. The results of this study demonstrate the ability of the organism to transform high concentrations of the oxidized form of CoQ(10) into the effective antioxidant (reduced) form and justify prospects of the development of parenteral dosage forms of CoQ(10) for the use in the treatment of acute pathological conditions. PMID:25762606

  10. Molecular diagnosis of coenzyme Q10 deficiency.

    PubMed

    Yubero, Delia; Montero, Raquel; Armstrong, Judith; Espinós, Carmen; Palau, Francesc; Santos-Ocaña, Carlos; Salviati, Leonardo; Navas, Placido; Artuch, Rafael

    2015-01-01

    Coenzyme Q10 (CoQ) deficiency syndromes comprise a growing number of neurological and extraneurological disorders. Primary-genetic but also secondary CoQ deficiencies have been reported. The biochemical determination of CoQ is a good tool for the rapid identification of CoQ deficiencies but does not allow the selection of candidate genes for molecular diagnosis. Moreover, the metabolic pathway for CoQ synthesis is an intricate and not well-understood process, where a large number of genes are implicated. Thus, only next-generation sequencing techniques (either genetic panels of whole-exome and -genome sequencing) are at present appropriate for a rapid and realistic molecular diagnosis of these syndromes. The potential treatability of CoQ deficiency strongly supports the necessity of a rapid molecular characterization of patients, since primary CoQ deficiencies may respond well to CoQ treatment. PMID:26144946

  11. Specificity and biological distribution of coenzyme M (2-mercaptoethanesulfonic acid).

    PubMed Central

    Balch, W E; Wolfe, R S

    1979-01-01

    The specificity of the growth requirement of Methanobacterium ruminantium strain M1 for a new coenzyme, 2-mercaptoethanesulfonic acid (HS--CoM), was examined. A variety of derivatives, analogs, and potential biosynthetic precursors of coenzyme M were tested; only a restricted range of thioether, thioester, and thiocarbonate derivatives of the cofactor were found to replace the HS--CoM requirement. Bromoethanesulfonic acid (BrCH2CH2SO3-), a halogenated analog of HS--CoM, potently inhibited the growth response. No coenzyme was detectable in a wide range of nonmethanogenic eucaryotic tissues and procaryotic organisms. However, all methanogens available in pure culture exhibited high levels of coenzyme M which ranged from 0.3 to 16 nmol/mg of dry weight. PMID:104960

  12. Neutron study of B 12 coenzyme at 15 K

    NASA Astrophysics Data System (ADS)

    Bouquiere, J. P.

    1992-06-01

    This high resolution and low temperature study, at 15 K, of the vitamin B 12 coenzyme ( C72H100N18O17PCo) was undertaken to confirm and clarify the water networks identified at 279 K by Savage [1]. Details of the data collection and refinement of the low temperature structure are described, a comparison of the coenzyme molecule structures at 15 and 279 K is made, and that of solvent structures outlined.

  13. Better than Nature: Nicotinamide Biomimetics That Outperform Natural Coenzymes

    PubMed Central

    2016-01-01

    The search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. Redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. Stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. Here, we investigate the efficiency of man-made synthetic biomimetics of the natural coenzymes NAD(P)H in redox biocatalysis. Extensive studies with a range of oxidoreductases belonging to the “ene” reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase XenA with selected biomimetics. In selected cases, these biomimetics outperform the natural coenzymes. “Better-than-Nature” biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost. PMID:26727612

  14. Plasma membrane coenzyme Q: evidence for a role in autism

    PubMed Central

    Crane, Frederick L; Löw, Hans; Sun, Iris; Navas, Placido; Gvozdjáková, Anna

    2014-01-01

    Background The Voltage Dependent Anion Channel (VDAC) is involved in control of autism. Treatments, including coenzyme Q, have had some success on autism control. Data sources Correlation of porin redox activity and expression of autism is based on extensive literature, especially studies of antibodies, identification of cytosolic nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase activity in the VDAC, and evidence for extreme sensitivity of the dehydrogenase to a mercurial. Evidence for a coenzyme Q requirement came from extraction and analog inhibition of NADH ferricyanide reductase in the erythrocyte plasma membrane, done in 1994, and reinterpreted when it was identified in VDAC in 2004. The effects of ubiquinol (the QH2 – reduced form of coenzyme Q) in children with autism were studied. Results A new role for coenzyme Q in the porin channels has implications on autism. Ubiquinol, the more active form of coenzyme Q, produces favorable response in children with autism. Agents which affected electron transport in porin show parallel effects in autism. Conclusion We propose a hypothesis that autism is controlled by a coenzyme Q-dependent redox system in the porin channels; this conclusion is based on the effects of agents that positively or negatively affect electron transport and the symptoms of autism. The full understanding of the mechanism of their control needs to be established. PMID:24920882

  15. Genetics of Coenzyme Q10 Deficiency

    PubMed Central

    Doimo, Mara; Desbats, Maria A.; Cerqua, Cristina; Cassina, Matteo; Trevisson, Eva; Salviati, Leonardo

    2014-01-01

    Coenzyme Q10 (CoQ10) is an essential component of eukaryotic cells and is involved in crucial biochemical reactions such as the production of ATP in the mitochondrial respiratory chain, the biosynthesis of pyrimidines, and the modulation of apoptosis. CoQ10 requires at least 13 genes for its biosynthesis. Mutations in these genes cause primary CoQ10 deficiency, a clinically and genetically heterogeneous disorder. To date mutations in 8 genes (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) have been associated with CoQ10 deficiency presenting with a wide variety of clinical manifestations. Onset can be at virtually any age, although pediatric forms are more common. Symptoms include those typical of respiratory chain disorders (encephalomyopathy, ataxia, lactic acidosis, deafness, retinitis pigmentosa, hypertrophic cardiomyopathy), but some (such as steroid-resistant nephrotic syndrome) are peculiar to this condition. The molecular bases of the clinical diversity of this condition are still unknown. It is of critical importance that physicians promptly recognize these disorders because most patients respond to oral administration of CoQ10. PMID:25126048

  16. Biosynthesis of coenzyme Q in eukaryotes.

    PubMed

    Kawamukai, Makoto

    2015-01-01

    Coenzyme Q (CoQ) is a component of the electron transport chain that participates in aerobic cellular respiration to produce ATP. In addition, CoQ acts as an electron acceptor in several enzymatic reactions involving oxidation-reduction. Biosynthesis of CoQ has been investigated mainly in Escherichia coli and Saccharomyces cerevisiae, and the findings have been extended to various higher organisms, including plants and humans. Analyses in yeast have contributed greatly to current understanding of human diseases related to CoQ biosynthesis. To date, human genetic disorders related to mutations in eight COQ biosynthetic genes have been reported. In addition, the crystal structures of a number of proteins involved in CoQ synthesis have been solved, including those of IspB, UbiA, UbiD, UbiX, UbiI, Alr8543 (Coq4 homolog), Coq5, ADCK3, and COQ9. Over the last decade, knowledge of CoQ biosynthesis has accumulated, and striking advances in related human genetic disorders and the crystal structure of proteins required for CoQ synthesis have been made. This review focuses on the biosynthesis of CoQ in eukaryotes, with some comparisons to the process in prokaryotes. PMID:26183239

  17. Coenzyme Q10 Deficiencies in Neuromuscular Diseases

    PubMed Central

    Salviati, Leonardo; Jackson, Sandra; Hirano, Michio; Navas, Plácido

    2011-01-01

    Coenzyme Q (CoQ) is an essential component of the respiratory chain but also participates in other mitochondrial functions such as regulation of the transition pore and uncoupling proteins. Furthermore, this compound is a specific substrate for enzymes of the fatty acids β–oxidation pathway and pyrimidine nucleotide biosynthesis. Furthermore, CoQ is an antioxidant that acts in all cellular membranes and lipoproteins. A complex of at least ten nuclear (COQ) genes encoded proteins synthesizes CoQ but its regulation is unknown. Since 1989, a growing number of patients with multisystemic mitochondrial disorders and neuromuscular disorders showing deficiencies of CoQ have been identified. CoQ deficiency caused by muta-tion(s) in any of the COQ genes is designated primary deficiency. Other patients have displayed other genetic defects independent on the CoQ biosynthesis pathway, and are considered to have secondary deficiencies. This review updates the clinical and molecular aspects of both types of CoQ deficiencies and proposes new approaches to understanding their molecular bases. PMID:20225022

  18. Clinical applications of coenzyme Q10.

    PubMed

    Garrido-Maraver, Juan; Cordero, Mario D; Oropesa-Avila, Manuel; Vega, Alejandro Fernandez; de la Mata, Mario; Pavon, Ana Delgado; Alcocer-Gomez, Elisabet; Calero, Carmen Perez; Paz, Marina Villanueva; Alanis, Macarena; de Lavera, Isabel; Cotan, David; Sanchez-Alcazar, Jose A

    2014-01-01

    Coenzyme Q10 (CoQ10) or ubiquinone was known for its key role in mitochondrial bioenergetics as electron and proton carrier; later studies demonstrated its presence in other cellular membranes and in blood plasma, and extensively investigated its antioxidant role. These two functions constitute the basis for supporting the clinical indication of CoQ10. Furthermore, recent data indicate that CoQ10 affects expression of genes involved in human cell signalling, metabolism and transport and some of the effects of CoQ10 supplementation may be due to this property. CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, ageing-related oxidative stress and carcinogenesis processes, and also a secondary effect of statin treatment. Many neurodegenerative disorders, diabetes, cancer, fibromyalgia, muscular and cardiovascular diseases have been associated with low CoQ10 levels. CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral CoQ10 treatment is a frequent mitochondrial energizer and antioxidant strategy in many diseases that may provide a significant symptomatic benefit. PMID:24389208

  19. Bioelectrochemical activity of an electroactive macromolecular weight coenzyme derivative

    NASA Astrophysics Data System (ADS)

    Liu, Pu; Zheng, Haitao; Nie, Pingping; Wei, Yaotian; Feng, Zhenchao; Sun, Tao

    2009-07-01

    As coenzyme utilized by more than hundreds of dehydrogenases, the efficient immobilization and regeneration of nicotinamide adenine dinucleotide (NAD+) are of great importance and have practical applications in industrial, analytical and biomedical field. In this paper, an electroactive macromolecular weight coenzyme derivative (PEI-DHBNAD) was prepared by attaching both NAD+ and 3,4-dihydroxybenzaldehyde (3,4-DHB) to a water-soluble polyelectrolyte, poly(ethylenimine) (PEI). The functional polymer exhibited both electrochemical properties of catechol unites and coenzymatic activity of NAD moieties. The macromolecular NAD analogue showed a substantial degree of efficiency relative to free NAD+ with alcohol dehydrogenase (ADH) and glucose-6-phophate dehydrogenase (G6PDH), and a litter higher Michaelis-Menton constant (Km) was obtained for the coenzyme derivative than free NAD+. The bioelectrochemical properties of PEI-DHB-NAD were investigated by using G6PDH as the model enzyme, and both of them were retained on electrode surface by ultrafiltration membrane. The modified electrode showed typical response to substrate without the addition of free coenzyme, which indicated that PEI-DHB-NAD can carry out the electron transfer between electrode and NAD-dependent dehydrogenase. The utilization of polymer-based PEI-DHB-NAD is convenient for the immobilization of both electron mediator and coenzyme, and offers a practical approach for the construction of reagentless biosensors.

  20. Coenzyme Q supplementation in pulmonary arterial hypertension

    PubMed Central

    Sharp, Jacqueline; Farha, Samar; Park, Margaret M.; Comhair, Suzy A.; Lundgrin, Erika L.; Tang, W.H. Wilson; Bongard, Robert D.; Merker, Marilyn P.; Erzurum, Serpil C.

    2014-01-01

    Mitochondrial dysfunction is a fundamental abnormality in the vascular endothelium and smooth muscle of patients with pulmonary arterial hypertension (PAH). Because coenzyme Q (CoQ) is essential for mitochondrial function and efficient oxygen utilization as the electron carrier in the inner mitochondrial membrane, we hypothesized that CoQ would improve mitochondrial function and benefit PAH patients. To test this, oxidized and reduced levels of CoQ, cardiac function by echocardiogram, mitochondrial functions of heme synthesis and cellular metabolism were evaluated in PAH patients (N=8) in comparison to healthy controls (N=7), at baseline and after 12 weeks oral CoQ supplementation. CoQ levels were similar among PAH and control individuals, and increased in all subjects with CoQ supplementation. PAH patients had higher CoQ levels than controls with supplementation, and a tendency to a higher reduced-to-oxidized CoQ ratio. Cardiac parameters improved with CoQ supplementation, although 6-minute walk distances and BNP levels did not significantly change. Consistent with improved mitochondrial synthetic function, hemoglobin increased and red cell distribution width (RDW) decreased in PAH patients with CoQ, while hemoglobin declined slightly and RDW did not change in healthy controls. In contrast, metabolic and redox parameters, including lactate, pyruvate and reduced or oxidized gluthathione, did not change in PAH patients with CoQ. In summary, CoQ improved hemoglobin and red cell maturation in PAH, but longer studies and/or higher doses with a randomized placebo-controlled controlled design are necessary to evaluate the clinical benefit of this simple nutritional supplement. PMID:25180165

  1. Coenzyme Q biosynthesis in health and disease.

    PubMed

    Acosta, Manuel Jesús; Vazquez Fonseca, Luis; Desbats, Maria Andrea; Cerqua, Cristina; Zordan, Roberta; Trevisson, Eva; Salviati, Leonardo

    2016-08-01

    Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:27060254

  2. Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis

    DOE PAGESBeta

    Sarmiento, Felipe; Ellison, Courtney K.; Whitman, William B.

    2013-01-01

    Coenzyme M is an essential coenzyme for methanogenesis. The proposed biosynthetic pathway consists of five steps, of which the fourth step is catalyzed by sulfopyruvate decarboxylase (ComDE). Disruption of the gene comE by transposon mutagenesis resulted in a partial coenzyme M auxotroph, which grew poorly in the absence of coenzyme M and retained less than 3% of the wild type level of coenzyme M biosynthesis. Upon coenzyme M addition, normal growth of the mutant was restored. Moreover, complementation of the mutation with the wild type comE gene in trans restored full growth in the absence of coenzyme M. Thesemore » results confirm that ComE plays an important role in coenzyme M biosynthesis. The inability to yield a complete CoM auxotroph suggests that either the transposon insertion failed to completely inactivate the gene or M. maripaludis possesses a promiscuous activity that partially complemented the mutation.« less

  3. Structural Insight into Methyl-Coenzyme M Reductase Chemistry Using Coenzyme B Analogues

    SciTech Connect

    Cedervall, Peder E.; Dey, Mishtu; Pearson, Arwen R.; Ragsdale, Stephen W.; Wilmot, Carrie M.

    2010-09-07

    Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F430. Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F430 nickel in the deepest part of the 30 {angstrom} long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 {angstrom} predominantly hydrophobic part of the channel close to F430, with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 {angstrom}. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB5SH, CoB6SH, CoB8SH, and CoB9SH, respectively). The data presented here reveal that the shorter CoB5SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB6SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB6SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB8SH and CoB9SH analogues can be accommodated in the active site through exclusion of this water. These analogues

  4. 3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate:coenzyme A ligase from cultured cells of Centaurium erythraea.

    PubMed

    Barillas, W; Beerhues, L

    1997-01-01

    3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth. PMID:9177055

  5. Coenzyme Q10 Administration Increases Brain Mitochondrial Concentrations and Exerts Neuroprotective Effects

    NASA Astrophysics Data System (ADS)

    Matthews, Russell T.; Yang, Lichuan; Browne, Susan; Baik, Myong; Flint Beal, M.

    1998-07-01

    Coenzyme Q10 is an essential cofactor of the electron transport chain as well as a potent free radical scavenger in lipid and mitochondrial membranes. Feeding with coenzyme Q10 increased cerebral cortex concentrations in 12- and 24-month-old rats. In 12-month-old rats administration of coenzyme Q10 resulted in significant increases in cerebral cortex mitochondrial concentrations of coenzyme Q10. Oral administration of coenzyme Q10 markedly attenuated striatal lesions produced by systemic administration of 3-nitropropionic acid and significantly increased life span in a transgenic mouse model of familial amyotrophic lateral sclerosis. These results show that oral administration of coenzyme Q10 increases both brain and brain mitochondrial concentrations. They provide further evidence that coenzyme Q10 can exert neuroprotective effects that might be useful in the treatment of neurodegenerative diseases.

  6. Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

    SciTech Connect

    Hawkins, AB; Adams, MWW; Kelly, RM

    2014-03-25

    The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73 degrees C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA beta-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.

  7. Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

    PubMed Central

    Hawkins, Aaron B.; Adams, Michael W. W.

    2014-01-01

    The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula. PMID

  8. Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions.

    PubMed

    Ko, Yeounjoo; Ashok, Somasundar; Ainala, Satish Kumar; Sankaranarayanan, Mugesh; Chun, Ah Yeong; Jung, Gyoo Yeol; Park, Sunghoon

    2014-12-01

    Coenzyme B12 (Vitamin B12 ) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B12 . These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B12 synthetic genes and successfully produced coenzyme B12 . However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B12 produced by the recombinant E. coli (0.21 ± 0.02 μg/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 ± 0.22 μg/g cdw). Optimization of the culture conditions to improve the production of coenzyme B12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 μg/g cdw) of coenzyme B12 was obtained. Interestingly, although the synthesis of coenzyme B12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B12 under anaerobic conditions. PMID:25146562

  9. Deregulated Coenzyme A, Loss of Metabolic Flexibility and Diabetes

    PubMed Central

    Jackowski, Suzanne; Leonardi, Roberta

    2016-01-01

    Coenzyme A (CoA) is an essential cofactor that is emerging as a global regulator of energy metabolism. Tissue CoA levels are tightly regulated and vary in response to different conditions including nutritional state and diabetes. Recent studies reveal the ability of this cofactor to control the output of key metabolic pathways. CoA regulation is important for the maintenance of metabolic flexibility and glucose homeostasis. PMID:25110012

  10. Coenzyme B12 Repurposed for Photoregulation of Gene Expression.

    PubMed

    Gruber, Karl; Kräutler, Bernhard

    2016-05-01

    Old cofactor, new tricks: In enzymes, coenzyme B12 has a well-known function as a radical initiator through homolysis of the Co-C bond. It has recently been shown that nature has repurposed this cofactor as a photosensitive switch for the regulation of bacterial carotenoid biosynthesis. Co-C bond breakage is again the key event in this process, triggering huge conformational changes in the B12 -binding protein. PMID:27010518

  11. Mitofusin 2 is required to maintain mitochondrial coenzyme Q levels

    PubMed Central

    Mourier, Arnaud; Motori, Elisa; Brandt, Tobias; Lagouge, Marie; Atanassov, Ilian; Galinier, Anne; Rappl, Gunter; Brodesser, Susanne; Hultenby, Kjell; Dieterich, Christoph

    2015-01-01

    Mitochondria form a dynamic network within the cell as a result of balanced fusion and fission. Despite the established role of mitofusins (MFN1 and MFN2) in mitochondrial fusion, only MFN2 has been associated with metabolic and neurodegenerative diseases, which suggests that MFN2 is needed to maintain mitochondrial energy metabolism. The molecular basis for the mitochondrial dysfunction encountered in the absence of MFN2 is not understood. Here we show that loss of MFN2 leads to impaired mitochondrial respiration and reduced ATP production, and that this defective oxidative phosphorylation process unexpectedly originates from a depletion of the mitochondrial coenzyme Q pool. Our study unravels an unexpected and novel role for MFN2 in maintenance of the terpenoid biosynthesis pathway, which is necessary for mitochondrial coenzyme Q biosynthesis. The reduced respiratory chain function in cells lacking MFN2 can be partially rescued by coenzyme Q10 supplementation, which suggests a possible therapeutic strategy for patients with diseases caused by mutations in the Mfn2 gene. PMID:25688136

  12. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

    PubMed Central

    Shieh, J; Whitman, W B

    1988-01-01

    To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation. PMID:3133359

  13. The role of zinc in the methylation of the coenzyme M thiol group in methanol:coenzyme M methyltransferase from Methanosarcina barkeri.

    PubMed

    Krüer, Markus; Haumann, Michael; Meyer-Klaucke, Wolfram; Thauer, Rudolf K; Dau, Holger

    2002-04-01

    Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237-->Ala and Cys239-->Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding. PMID:11985589

  14. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

    PubMed Central

    Angelin, B

    1988-01-01

    The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis. PMID:3202831

  15. Molecular Insights into the Biosynthesis of the F420 Coenzyme

    SciTech Connect

    Forouhar,F.; Abashidze, M.; Xu, H.; Grochowski, L.; Seetharaman, J.; Hussain, M.; Kuzin, A.; Chen, Y.; Zhou, W.; et al

    2008-01-01

    Coenzyme F420, a hydride carrier, is found in Archaea and some bacteria and has crucial roles in methanogenesis, antibiotic biosynthesis, DNA repair, and activation of antitubercular compounds. CofD, 2-phospho-l-lactate transferase, catalyzes the last step in the biosynthesis of F420-0 (F420 without polyglutamate), by transferring the lactyl phosphate moiety of lactyl(2)diphospho-(5')guanosine to 7,8-didemethyl-8-hydroxy-5-deazariboflavin ribitol (Fo). CofD is highly conserved among F420-producing organisms, and weak sequence homologs are also found in non-F420-producing organisms. This superfamily does not share any recognizable sequence conservation with other proteins. Here we report the first crystal structures of CofD, the free enzyme and two ternary complexes, with Fo and Pi or with Fo and GDP, from Methanosarcina mazei. The active site is located at the C-terminal end of a Rossmann fold core, and three large insertions make significant contributions to the active site and dimer formation. The observed binding modes of Fo and GDP can explain known biochemical properties of CofD and are also supported by our binding assays. The structures provide significant molecular insights into the biosynthesis of the F420 coenzyme. Large structural differences in the active site region of the non-F420-producing CofD homologs suggest that they catalyze a different biochemical reaction.

  16. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%. PMID:26856529

  17. Excited flavin and pterin coenzyme molecules in evolution.

    PubMed

    Kritsky, M S; Telegina, T A; Vechtomova, Y L; Kolesnikov, M P; Lyudnikova, T A; Golub, O A

    2010-10-01

    Excited flavin and pterin molecules are active in intermolecular energy transfer and in photocatalysis of redox reactions resulting in conservation of free energy. Flavin-containing pigments produced in models of the prebiotic environment are capable of converting photon energy into the energy of phosphoanhydride bonds of ATP. However, during evolution photochemical reactions involving excited FMN or FAD molecules failed to become participants of bioenergy transfer systems, but they appear in enzymes responsible for repair of UV-damaged DNA (DNA photolyases) and also in receptors of blue and UV-A light regulating vital functions of organisms. The families of these photoproteins (DNA-photolyases and cryptochromes, LOV-domain- and BLUF-domain-containing proteins) are different in the structure and in mechanisms of the photoprocesses. The excited flavin molecules are involved in photochemical processes in reaction centers of these photoproteins. In DNA photolyases and cryptochromes the excitation energy on the reaction center flavin is supplied from an antenna molecule that is bound with the same polypeptide. The role of antenna is played by MTHF or by 8-HDF in some DNA photolyases, i.e. also by molecules with known coenzyme functions in biocatalysis. Differences in the structure of chromophore-binding domains suggest an independent origin of the photoprotein families. The analysis of structure and properties of coenzyme molecules reveals some specific features that were significant in evolution for their being selected as chromophores in these proteins. PMID:21166638

  18. Coenzyme Q-10 in Human Health: Supporting Evidence?

    PubMed

    Saha, Sibu P; Whayne, Thomas F

    2016-01-01

    Coenzyme Q-10 (CoQ10) is a widely used alternative medication or dietary supplement and one of its roles is as an antioxidant. It naturally functions as a coenzyme and component of oxidative phosphorylation in mitochondria. Decreased levels have been demonstrated in diseased myocardium and in Parkinson disease. Farnesyl pyrophosphate is a critical intermediate for CoQ10 synthesis and blockage of this step may be important in statin myopathy. Deficiency of CoQ10 also has been associated with encephalomyopathy, severe infantile multisystemic disease, cerebellar ataxia, nephrotic syndrome, and isolated myopathy. Although supplementation with CoQ10 has been reported to be beneficial in treating hypertension, congestive heart failure, statin myopathy, and problems associated with chemotherapy for cancer treatement, this use of CoQ10 as a supplement has not been confirmed in randomized controlled clinical trials. Nevertheless, it appears to be a safe supplementary medication where usage in selected clinical situations may not be inappropriate. This review is an attempt to actualize the available information on CoQ10 and define its potential benefit and appropriate usage. PMID:26741866

  19. Preparation and physicochemical characterization of aqueous dispersions of coenzyme Q10 nanoparticles.

    PubMed

    Siekmann, B; Westesen, K

    1995-02-01

    The present study describes a novel pharmaceutical formulation of coenzyme Q10, viz. submicron-sized dispersions of the substance prepared by emulsification of molten coenzyme Q10 in an aqueous phase. Photon correlation spectroscopy reveals mean diameters of 60 to 300 nm depending on process parameters. Coenzyme Q10 nanoparticles remain stable on storage for more than 30 months. Lipophilic drugs can be incorporated into the nanoparticles demonstrating their potential use as a drug carrier system. Transmission electron micrographs of freeze-fractured replica show spherical particles with an amorphous core. Cryo-electron microscopy reveals the coexistence of small unilamellar vesicles in phospholipid stabilized dispersions. Thermoanalysis and X-ray studies indicate that the dispersed and emulsified coenzyme Q10 does not recrystallize even at 4 degrees C over 30 months. These agree with 1H NMR data which demonstrate that coenzyme Q10 molecules have a high mobility when formulated as nanoparticles and that colloidally dispersed coenzyme Q10 remains in the state of a supercooled melt. Despite the high melting point of the bulk material, coenzyme Q10 dispersions represent no suspensions but O/W emulsions according to the IUPAC definition (1). PMID:7784334

  20. 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. PMID:26743849

  1. Synthetic biology for engineering acetyl coenzyme A metabolism in yeast.

    PubMed

    Nielsen, Jens

    2014-01-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol. PMID:25370498

  2. Pyridine nucleotide coenzymes: Chemical, biological, and medical aspects. Vol. 2, Pt. A

    SciTech Connect

    Dolphin, D.; Poulson, R.; Avramovic, O.

    1987-01-01

    This text contains the following: History of the Pyridine Nucleotides Nomenclature; Evolution of Pyridine Nucleotide; Relationship Between Biosynthesis and Evolution; Crystal Structure; Coenzyme Conformations; Protein Interactions; Optical Spectroscopy of the Pyridine Nucleotides; Excited States of Pyridine Nucleotide Coenzymes; Fluorescence and Phosphorescence; Nuclear Magnetic Resonance Spectroscopy of Pyridine Nucleotides; Mass Spectrometry of Pyridine Nucleotides; Mechanism of Action of the Pyridine Nucleotides; Chemical Stability and Reactivity of Pyridine Nucleotide Coenzymes; Stereochemistry of Fatty Acid Biosynthesis and Metabolism; Kinetics of Pyridine Nucleotide-Utilizing Enzymes; Preparation and Properties of NAD and NADP Analogs; Model Studies and Biological Activity of Analogs; and Spin-Labeled Pyridine Nucleotide Derivatives.

  3. Tissue concentrations of coenzyme Q10 in the rat following its oral and intraperitoneal administration.

    PubMed

    Reahal, S; Wrigglesworth, J

    1992-01-01

    Daily oral or ip administration of coenzyme Q10 to rats for time periods of 2 to 10 weeks leads to its accumulation in liver, concentrating in the soluble fraction of the liver cells. No uptake of coenzyme Q10 can be detected in the heart or kidney. Intraperitoneal administration also results in the accumulation of coenzyme Q10 in the spleen. It is concluded that the normal endogenous levels of quinone in the rat heart and kidney cannot be supplemented over the long term by administration of exogenous quinone. PMID:1355718

  4. Coenzyme world model of the origin of life.

    PubMed

    Sharov, Alexei A

    2016-06-01

    The origin of life means the emergence of heritable and evolvable self-reproduction. However the mechanisms of primordial heredity were different from those in contemporary cells. Here I argue that primordial life had no nucleic acids; instead heritable signs were represented by isolated catalytically active self-reproducing molecules, similar to extant coenzymes, which presumably colonized surfaces of oil droplets in water. The model further assumes that coenzyme-like molecules (CLMs) changed surface properties of oil droplets (e.g., by oxidizing terminal carbons), and in this way created and sustained favorable conditions for their own self-reproduction. Such niche-dependent self-reproduction is a necessary condition for cooperation between different kinds of CLMs because they have to coexist in the same oil droplets and either succeed or perish together. Additional kinds of hereditary molecules were acquired via coalescence of oil droplets carrying different kinds of CLMs or via modification of already existing CLMs. Eventually, polymerization of CLMs became controlled by other polymers used as templates; and this kind of template-based synthesis eventually resulted in the emergence of RNA-like replicons. Apparently, oil droplets transformed into the outer membrane of cells via engulfing water, stabilization of the surface, and osmoregulation. In result, the metabolism was internalized allowing cells to accumulate free-floating resources (e.g., animoacids, ATP), which was a necessary condition for the development of protein synthesis. Thus, life originated from simple but already functional molecules, and its gradual evolution towards higher complexity was driven by cooperation and natural selection. PMID:26968100

  5. The Reaction Mechanism of Methyl-Coenzyme M Reductase

    PubMed Central

    Wongnate, Thanyaporn; Ragsdale, Stephen W.

    2015-01-01

    Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mm). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(NiI)·CH3SCoM) is highly favored (Kd = 79 μm). Only then can the chemical reaction occur (kobs = 20 s−1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII)·CoB7S−·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates. PMID:25691570

  6. Component A of the methyl coenzyme M methylreductase system of Methanobacterium: resolution into four components.

    PubMed Central

    Nagle, D P; Wolfe, R S

    1983-01-01

    Component A, the oxygen-sensitive protein fraction of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum, has been stabilized and resolved into three protein fractions and one cofactor that are required to reconstitute component A activity. Component A1 is oxygen-stable and contains hydrogen-dependent deazaflavin (coenzyme F420)-reducing activity. Component A2 is acidic; components A2 and A3 are oxygen sensitive. The specific functions of each component in methyl group reduction are unknown. Resolution of component A revealed a new cofactor requirement of the methylreductase system for FAD. Hydrogen-dependent reduction of methyl coenzyme M to methane and coenzyme M, the terminal step of CO2 reduction by methanogenic bacteria, requires protein components A1, A2, A3, and C in addition to component B, FAD, ATP, and Mg2+. PMID:6403944

  7. Supplementation of Coenzyme Q10 among Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Shen, Qiuhua; Pierce, Janet D.

    2015-01-01

    Type 2 diabetes mellitus (T2DM) is a major cause of morbidity and mortality with ever increasing prevalence in the United States and worldwide. There is growing body of evidence suggesting that mitochondrial dysfunction secondary to oxidative stress plays a critical role in the pathogenesis of T2DM. Coenzyme Q10 is an important micronutrient acting on the electron transport chain of the mitochondria with two major functions: (1) synthesis of adenosine triphosphate (ATP); and (2) a potent antioxidant. Deficiency in coenzyme Q10 is often seen in patients with T2DM. Whether restoration of coenzyme Q10 will help alleviate oxidative stress, preserve mitochondrial function, and thus improve glycemic control in T2DM is unclear. This article reviews the relationships among oxidative stress, mitochondrial dysfunction, and T2DM and examines the evidence for potential use of coenzyme Q10 as a supplement for the treatment of T2DM. PMID:27417763

  8. Coenzyme Q10 and periodontal treatment: is there any beneficial effect?

    PubMed

    Watts, T L

    1995-03-25

    Many dentists have been surprised by recent media claims of periodontal benefits with a purportedly revolutionary dietary supplement. The research literature on coenzyme Q10's periodontal effects does not extend to the international English language dental literature, which perhaps explains the surprise. A review of the available literature does not give any ground for the claims made, and selected papers are discussed to show that there is actually some evidence that coenzyme Q10 has no place in periodontal treatment. PMID:7718355

  9. Purification and Characterization of Cinnamoyl-Coenzyme A:NADP Oxidoreductase in Eucalyptus gunnii.

    PubMed Central

    Goffner, D.; Campbell, M. M.; Campargue, C.; Clastre, M.; Borderies, G.; Boudet, A.; Boudet, A. M.

    1994-01-01

    Cinnamoyl-coenzyme A:NADP oxidoreductase (CCR, EC 1.2.1.44), the entry-point enzyme into the monolignol biosynthetic pathway, was purified to apparent electrophoretic homogeneity from differentiating xylem of Eucalyptus gunnii Hook. The purified protein is a monomer of 38 kD and has an isoelectric point of 7. Although Eucalyptus gunnii CCR has approximately equal affinities for all possible substrates (p-coumaroyl-coenzyme A, feruloyl-coenzyme A, and sinapoyl-coenzyme A), it is approximately three times more effective at converting feruloyl-coenzyme A than the other substrates. To gain a better understanding of the catalytic regulation of Eucalyptus CCR, a variety of compounds were tested to determine their effect on CCR activity. CCR activity is inhibited by NADP and coenzyme A. Effectors that bind lysine and cysteine residues also inhibit CCR activity. As a prerequisite to the study of the regulation of CCR at the molecular level, polyclonal antibodies were obtained. PMID:12232355

  10. The Relationship between Coenzyme Q10, Oxidative Stress, and Antioxidant Enzymes Activities and Coronary Artery Disease

    PubMed Central

    Lee, Bor-Jen; Lin, Yi-Chin; Huang, Yi-Chia; Ko, Ya-Wen; Hsia, Simon; Lin, Ping-Ting

    2012-01-01

    A higher oxidative stress may contribute to the pathogenesis of coronary artery disease (CAD). The purpose of this study was to investigate the relationship between coenzyme Q10 concentration and lipid peroxidation, antioxidant enzymes activities and the risk of CAD. Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery were assigned to the case group (n = 51). The control group (n = 102) comprised healthy individuals with normal blood biochemical values. The plasma coenzyme Q10, malondialdehyde (MDA) and antioxidant enzymes activities (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx)) were measured. Subjects with CAD had significant lower plasma coenzyme Q10, CAT and GPx activities and higher MDA and SOD levels compared to those of the control group. The plasma coenzyme Q10 was positively correlated with CAT and GPx activities and negatively correlated with MDA and SOD. However, the correlations were not significant after adjusting for the potential confounders of CAD with the exception of SOD. A higher level of plasma coenzyme Q10 (≥0.52 μmol/L) was significantly associated with reducing the risk of CAD. Our results support the potential cardioprotective impact of coenzyme Q10. PMID:22645453

  11. Decreased Coenzyme Q10 Levels in Multiple System Atrophy Cerebellum.

    PubMed

    Barca, Emanuele; Kleiner, Giulio; Tang, Guomei; Ziosi, Marcello; Tadesse, Saba; Masliah, Eliezer; Louis, Elan D; Faust, Phyllis; Kang, Un J; Torres, Jose; Cortes, Etty P; Vonsattel, Jean-Paul G; Kuo, Sheng-Han; Quinzii, Catarina M

    2016-07-01

    In familial and sporadic multiple system atrophy (MSA) patients, deficiency of coenzyme Q10 (CoQ10) has been associated with mutations in COQ2, which encodes the second enzyme in the CoQ10 biosynthetic pathway. Cerebellar ataxia is the most common presentation of CoQ10 deficiency, suggesting that the cerebellum might be selectively vulnerable to low levels of CoQ10 To investigate whether CoQ10 deficiency represents a common feature in the brains of MSA patients independent of the presence of COQ2 mutations, we studied CoQ10 levels in postmortem brains of 12 MSA, 9 Parkinson disease (PD), 9 essential tremor (ET) patients, and 12 controls. We also assessed mitochondrial respiratory chain enzyme activities, oxidative stress, mitochondrial mass, and levels of enzymes involved in CoQ biosynthesis. Our studies revealed CoQ10 deficiency in MSA cerebellum, which was associated with impaired CoQ biosynthesis and increased oxidative stress in the absence of COQ2 mutations. The levels of CoQ10 in the cerebella of ET and PD patients were comparable or higher than in controls. These findings suggest that CoQ10 deficiency may contribute to the pathogenesis of MSA. Because no disease modifying therapies are currently available, increasing CoQ10 levels by supplementation or upregulation of its biosynthesis may represent a novel treatment strategy for MSA patients. PMID:27235405

  12. The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

    PubMed Central

    Lawrence, J G; Roth, J R

    1995-01-01

    The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria. PMID:7592411

  13. Neutron Laue diffraction studies of coenzyme cob(II)alamin.

    PubMed

    Langan, P; Lehmann, M; Wilkinson, C; Jogl, G; Kratky, C

    1999-01-01

    Using a recently designed neutron single-crystal diffractometer utilizing a narrow-band Laue concept (LADI), diffraction data were collected from a crystal of the coenzyme cob(II)alamin (B12r), crystallized from a mixture of D2O and perdeuterated acetone. The instrument was placed at the end of a cold neutron guide at the Institute Laue Langevin (ILL, Grenoble, France), and data collection with neutrons of 1.8-8.0 A wavelength to a crystallographic resolution of 1.43 A was complete after about 36 h. This compares favourably with a previous experiment utilizing the same crystal specimen, where more than four weeks were required to collect monochromatic diffraction data to about 1 A resolution. Using the Laue data, the structure was solved by molecular replacement with the known X-ray crystal structure. Difference density maps revealed the atomic positions (including deuterium atoms) of seven ordered solvent water molecules and two (partially disordered) acetone molecules. These density maps were compared with corresponding maps computed with monochromatic neutron-diffraction data collected to 1. 0 A resolution using the same crystal specimen, as well as to maps derived from high-resolution (0.90 A) synchrotron X-ray data. In spite of the better definition of atomic positions in the two high-resolution maps, the 1.43 A LADI maps show considerable power for the determination of the location of hydrogen and deuterium positions. PMID:10089394

  14. Coenzyme Q10 analytical determination in biological matrices and pharmaceuticals.

    PubMed

    Lucangioli, Silvia; Martinefski, Manuela; Tripodi, Valeria

    2016-01-01

    In recent years, the analytical determination of coenzyme Q10 (CoQ10) has gained importance in clinical diagnosis and in pharmaceutical quality control. CoQ10 is an important cofactor in the mitochondrial respiratory chain and a potent endogenous antioxidant. CoQ10 deficiency is often associated with numerous diseases and patients with these conditions may benefit from administration of supplements of CoQ10. In this regard, it has been observed that the best benefits are obtained when CoQ10 deficiency is diagnosed and treated early. Therefore, it is of great value to develop analytical methods for the detection and quantification of CoQ10 in this type of disease. The methods above mentioned should be simple enough to be used in routine clinical laboratories as well as in quality control of pharmaceutical formulations containing CoQ10. Here, we discuss the advantages and disadvantages of different methods of CoQ10 analysis. PMID:27100710

  15. Protein Acetylation and Acetyl Coenzyme A Metabolism in Budding Yeast

    PubMed Central

    Galdieri, Luciano; Zhang, Tiantian; Rogerson, Daniella; Lleshi, Rron

    2014-01-01

    Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation. PMID:25326522

  16. Coenzyme Q10 protects hair cells against aminoglycoside.

    PubMed

    Sugahara, Kazuma; Hirose, Yoshinobu; Mikuriya, Takefumi; Hashimoto, Makoto; Kanagawa, Eiju; Hara, Hirotaka; Shimogori, Hiroaki; Yamashita, Hiroshi

    2014-01-01

    It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10) is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group). In the neomycin group, utricles were cultured with neomycin (1 mM) to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30-0.3 µM). Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear. PMID:25265538

  17. MeaA, a Putative Coenzyme B12-Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

    PubMed Central

    Zhang, Weiwen; Reynolds, Kevin A.

    2001-01-01

    The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE∗ promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis

  18. Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei.

    PubMed Central

    Peck, M W

    1989-01-01

    Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant. PMID:2729992

  19. Coenzyme Q10 protects ischemic myocardium in an open-chest swine model.

    PubMed

    Atar, D; Mortensen, S A; Flachs, H; Herzog, W R

    1993-01-01

    Myocardial stunning, defined as a reversible decrease in contractility after ischemia and reperfusion, may be a manifestation of reperfusion injury caused by free oxygen radical damage. The aim of this study was to test the hypothesis that pretreatment with coenzyme Q10 (ubiquinone), believed to act as a free radical scavenger, reduces myocardial stunning in a porcine model. Twelve swine were randomized to receive either oral supplementation with coenzyme Q10 or placebo for 20 days. A normothermic open-chest model was used with short occlusion (8 min) of the distal left descending coronary artery followed by reperfusion. Regional contractile function was measured with epicardial Doppler crystals in ischemic and nonischemic segments by measuring thickening fraction of the left ventricular wall during systole. Stunning time was defined as the elapsed time of reduced contractility until return to baseline. Coenzyme Q10 concentrations were measured in blood and homogenized myocardial tissue by high performance liquid chromatography. Plasma levels of reduced coenzyme Q10 (ubiquinol) were higher in swine pretreated with the experimental medication as compared to placebo (mean 0.45 mg/l versus 0.11 mg/l, respectively). Myocardial tissue concentrations, however, did not show any changes (mean 0.79 micrograms/mg dry weight versus 0.74 micrograms/mg). Stunning time was significantly reduced in coenzyme Q10 pretreated animals (13.7 +/- 7.7 min versus 32.8 +/- 3.1 min, P < 0.01). In conclusion, chronic pretreatment with coenzyme Q10 protects ischemic myocardium in an open-chest swine model. The beneficial effect of coenzyme Q10 on myocardial stunning may be due to protection from free radical mediated reperfusion injury. This protective effect seems to be generated by a humoral rather than intracellular mechanism. PMID:8241692

  20. Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Alber, Birgit E; Fuchs, Georg

    2006-09-01

    The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route. PMID:16952935

  1. The Antioxidant Status and Concentrations of Coenzyme Q10 and Vitamin E in Metabolic Syndrome

    PubMed Central

    Yen, Chi-Hua; Yang, Nae-Cherng; Lee, Bor-Jen; Lin, Jui-Yuan; Hsia, Simon

    2013-01-01

    The purpose of this study was to investigate the levels of coenzyme Q10 and vitamin E and the antioxidant status in subjects with metabolic syndrome (MS). Subjects with MS (n = 72) were included according to the criteria for MS. The non-MS group (n = 105) was comprised of healthy individuals with normal blood biochemical values. The plasma coenzyme Q10, vitamin E concentrations, lipid profiles, and antioxidant enzymes levels (catalase, superoxide dismutase, and glutathione peroxidase) were measured. The subjects with MS had significantly higher concentrations of plasma coenzyme Q10 and vitamin E than those in the non-MS group, but these differences were not significant after being normalized for triglyceride level. The levels of antioxidant enzymes were significantly lower in the MS group than in the non-MS group. The subjects with the higher antioxidant enzymes activities had significant reductions in the risk of MS (P < 0.01) after being adjusted for coenzyme Q10 and vitamin E. In conclusion, the subjects with MS might be under higher oxidative stress resulting in low levels of antioxidant enzyme activities. A higher level of antioxidant enzymes activities was significantly associated with a reduction in the risk of MS independent of the levels of coenzyme Q10 and vitamin E. PMID:24082857

  2. Nutritional programming of coenzyme Q: potential for prevention and intervention?

    PubMed

    Tarry-Adkins, Jane L; Fernandez-Twinn, Denise S; Chen, Jian-Hua; Hargreaves, Iain P; Martin-Gronert, Malgorzata S; McConnell, Josie M; Ozanne, Susan E

    2014-12-01

    Low birth weight and rapid postnatal growth increases risk of cardiovascular-disease (CVD); however, underlying mechanisms are poorly understood. Previously, we demonstrated that rats exposed to a low-protein diet in utero that underwent postnatal catch-up growth (recuperated) have a programmed deficit in cardiac coenzyme Q (CoQ) that was associated with accelerated cardiac aging. It is unknown whether this deficit occurs in all tissues, including those that are clinically accessible. We investigated whether aortic and white blood cell (WBC) CoQ is programmed by suboptimal early nutrition and whether postweaning dietary supplementation with CoQ could prevent programmed accelerated aging. Recuperated male rats had reduced aortic CoQ [22 d (35±8.4%; P<0.05); 12 m (53±8.8%; P<0.05)], accelerated aortic telomere shortening (P<0.01), increased DNA damage (79±13% increase in nei-endonucleaseVIII-like-1), increased oxidative stress (458±67% increase in NAPDH-oxidase-4; P<0.001), and decreased mitochondrial complex II-III activity (P<0.05). Postweaning dietary supplementation with CoQ prevented these detrimental programming effects. Recuperated WBCs also had reduced CoQ (74±5.8%; P<0.05). Notably, WBC CoQ levels correlated with aortic telomere-length (P<0.0001) suggesting its potential as a diagnostic marker of vascular aging. We conclude that early intervention with CoQ in at-risk individuals may be a cost-effective and safe way of reducing the global burden of CVDs. PMID:25172893

  3. Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

    PubMed

    Zallot, Rémi; Agrimi, Gennaro; Lerma-Ortiz, Claudia; Teresinski, Howard J; Frelin, Océane; Ellens, Kenneth W; Castegna, Alessandra; Russo, Annamaria; de Crécy-Lagard, Valérie; Mullen, Robert T; Palmieri, Ferdinando; Hanson, Andrew D

    2013-06-01

    Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes. PMID:23590975

  4. Reduction of ascites mortality in broilers by coenzyme Q10.

    PubMed

    Geng, A L; Guo, Y M; Yang, Y

    2004-09-01

    Effects of coenzyme Q10 (CoQ10) supplementation on growth performance and ascites were studied in broilers. One hundred eighty 1-d-old Arbor Acre male broiler chicks were randomly allocated into 3 groups with 6 replicates each. From d 8, the diets were supplemented with CoQ10 at levels of 0, 20, and 40 mg/kg, respectively. From d 15 to 21, all the chicks were exposed to low ambient temperature (15 to 18 degrees C) to induce ascites. Average feed intake, BW gain, and feed conversion ratio of the broilers during 0 to 3 wk, 3 to 6 wk, and 0 to 6 wk were measured. The results showed that there were no influences observed on broilers' growth performance, but the mortality due to ascites was reduced by CoQ10 supplementation (P < or = 0.05). Erythrocyte osmotic fragility (EOF) was significantly decreased by 40 mg/kg CoQ10 compared with the control, but no significant changes were observed on blood packed cell volume (PCV) among the treatments. Pulmonary arterial diastolic pressure was significantly decreased on d 36, but no significant changes were observed on right ventricular pressure (RVP), pulmonary arterial systolic pressure, and the maximum change ratio of right intraventricular pressure (+/- dp/ dtmax). Ascites heart index (AHI) was significantly decreased by 40 mg/kg CoQ10 supplementation (P < or = 0.05). The results of this study suggested that CoQ10 has a beneficial effect in reducing ascites mortality in broilers, and 40 mg/kg CoQ10 seems to be more effective than 20 mg/ kg CoQ10. PMID:15384911

  5. Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Human Fibroblasts by Lipoproteins

    PubMed Central

    Brown, Michael S.; Dana, Suzanna E.; Goldstein, Joseph L.

    1973-01-01

    The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of hepatic cholesterol biosynthesis, is suppressed in human fibroblasts cultured in the presence of serum. This enzyme activity increases by more than 10-fold after the removal of serum from the medium. The rise in enzyme activity requires de novo protein synthesis and is not accompanied by changes in the activities of several other cellular enzymes. The factor responsible for the suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts is present in the sera of at least four mammalian species, and in human serum it is found in the low-density lipoproteins. Human high-density lipoproteins, very low-density lipoproteins from chicken egg yolk, and the fraction of human serum containing no lipoproteins do not suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. PMID:4352976

  6. The effect of simvastatin on coenzyme Q and antioxidant/oxidant balance in diabetic-hypercholesterolaemic rats.

    PubMed

    Kuzelová, Magdaléna; Adameová, Adriana; Sumbalová, Zuzana; Paulíková, Ingrid; Harcárová, Anna; Svec, Pavel; Kucharská, Jarmila

    2008-12-01

    The effect of simvastatin administered for 10 days on coenzyme Q and antioxidant/oxidant balance in a rat model of diabetes mellitus and hypercholesterolaemia was studied. In the diabetic-hypercholesterolaemic rats the signs of oxidative stress-decreased alpha-tocopherol/cholesterol in the plasma (p < 0.01) and alpha-tocopherol in liver (p < 0.001) together with increased lipid peroxidation in the liver (TBARS, p < 0.05) were found. Increased coenzyme Q9 concentrations in the plasma (p < 0.05) and liver (p < 0.01), coenzyme Q10 in the myocardium (p < 0.05) and in the liver (p < 0.01) may indicate adaptation to oxidative stress. Administration of simvastatin (10 mg/kg) to the diabetic-hypercholesterolaemic rats counteracted increased myocardial (coenzyme Q10, p < 0.05) and liver (total coenzyme Q9, p < 0.05) coenzyme Q concentrations but did not improve alpha-tocopherol depletion and increased formation of TBARS in the liver. Even though simvastatin treatment did not induce coenzyme Q deficiency in plasma, heart and liver of the diabetic-hypercholesterolaemic rats as compared to the control levels, it was not able to prevent completely the changes in antioxidant/oxidant balance induced by diabetes and hypercholesterolaemia. The results highlight the importance of studying the effect of statins on the coenzyme Q levels in the animal models of pathological conditions known to change the initial antioxidant defence system. PMID:19202203

  7. Effects of fluvastatin and coenzyme Q10 on skeletal muscle in normo- and hypercholesterolaemic rats.

    PubMed

    Vincze, J; Jenes, Á; Füzi, M; Almássy, J; Németh, R; Szigeti, G; Dienes, B; Gaál, Z; Szentesi, P; Jóna, I; Kertai, P; Paragh, G; Csernoch, L

    2015-06-01

    Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition. PMID:25920381

  8. Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

    PubMed Central

    Bokranz, M; Bäumner, G; Allmansberger, R; Ankel-Fuchs, D; Klein, A

    1988-01-01

    The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes. Images PMID:2448287

  9. Estimation of Methanogen Biomass by Quantitation of Coenzyme M

    PubMed Central

    Elias, Dwayne A.; Krumholz, Lee R.; Tanner, Ralph S.; Suflita, Joseph M.

    1999-01-01

    Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363–370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 ± 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 ± 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass. PMID:10584015

  10. Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

    PubMed Central

    Chee-Sanford, J C; Frost, J W; Fries, M R; Zhou, J; Tiedje, J M

    1996-01-01

    A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after

  11. Nano-encapsulation of coenzyme Q10 using octenyl succinic anhydride modified starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Octenyl succinic anhydride modified starch (OSA-ST) was used to encapsulate Coenzyme Q10 (CoQ10). CoQ10 was dissolved in rice bran oil (RBO), and incorporated into an aqueous OSA-ST solution. High pressure homogenization (HPH) of the mixture was conducted at 170 MPa for 5-6 cycles. The resulting ...

  12. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  13. Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans.

    PubMed

    Yin, Bo; Cui, Dongbing; Zhang, Lujia; Jiang, Shuiqin; Machida, Satoru; Yuan, Y Adam; Wei, Dongzhi

    2014-11-01

    Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets. PMID:24825769

  14. A possible prebiotic synthesis of pantetheine, a precursor to coenzyme A

    NASA Technical Reports Server (NTRS)

    Keefe, A. D.; Newton, G. L.; Miller, S. L.

    1995-01-01

    The involvement of coenzyme A in many enzyme reactions suggests that it acted in this capacity very early in the development of life on Earth. Particularly relevant in this regard is its role in the activation of amino acids and hydroxy acids in the biosynthesis of some peptide antibiotics--a mechanism of peptide synthesis that forms the basis for the proposal that a thioester world could have preceded the RNA world. The components of coenzyme A have been shown to be probable prebiotic compounds: beta-alanine, pantoyl lactone and cysteamine and possibly adenosine. We show here that the pantetheine moiety of coenzyme A (which also occurs in a number of enzymes) can be synthesized in yields of several per cent by heating pantoyl lactone, beta-alanine and cysteamine at temperatures as low as 40 degrees C. These components are extremely soluble and so would have been preferentially concentrated in evaporating bodies of water, for example on beaches and at lagoon margins. Our results show that amide bonds can be formed at temperatures as low as 40 degrees C, and provide circumstantial support for the suggestion that pantetheine and coenzyme A were important in the earliest metabolic systems.

  15. A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.

    PubMed

    Furuya, Toshiki; Miura, Misa; Kino, Kuniki

    2014-10-13

    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics. PMID:25164030

  16. 4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

    PubMed Central

    Gibson, J; Dispensa, M; Fogg, G C; Evans, D T; Harwood, C S

    1994-01-01

    Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound. Images PMID:8300518

  17. Characterization of the enzymatic conversion of sulfoacetaldehyde and L-cysteine into coenzyme M (2-mercaptoethanesulfonic acid)

    SciTech Connect

    White, R.H. )

    1988-09-20

    Sulfoacetaldehyde was shown to be converted enzymatically into coenzyme M by cell-free extracts of methanogenic bacteria. Gas chromatography-mass spectrometry (GC-MS) of the S-methyl methyl ester derivative of the coenzyme M isolated from the extracts was used to measure both the extent and position of the deuterium incorporated into coenzyme M from (2,2-{sup 2}H{sub 2})sulfoacetaldehyde. The conversion of sulfoacetaldehyde into coenzyme M was greatly stimulated by the addition of L-cysteine (20 mM) to the extracts and/or by incubating the extracts under hydrogen, whereas incubation in the presence of sulfide (20 mM) greatly reduced coenzyme M synthesis. Incubation of a cell-free extract from Methanobacterium formicicum with (2,2-{sup 2}H{sub 2})sulfoacetaldehyde and ({sup 34}S)-L-cysteine (92.6 atom % {sup 34}S) led to the production of coenzyme M in which the thiol portion of the molecule contained 90 atom % {sup 34}S. (ethylene-{sup 2}H{sub 4})-S-(2-Sulfoethyl)cysteine, incubated with this cell-free extract at a concentration of 22 mM, readily cleaved to coenzyme M. On the basis of these observations, it is concluded that sulfoacetaldehyde is converted into coenzyme M by reacting with cysteine to form the thiazolidine adduct (2-(sulfomethyl)thiazolidine-4-carboxylic acid), which undergoes a reductive cleavage of the heterocyclic C(2)-N bond to form S-(2-sulfoethyl)cysteine, which, in turn, undergoes a {beta}-elimination to produce coenzyme M.

  18. Benzoate-coenzyme A ligase, encoded by badA, is one of three ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth of Rhodopseudomonas palustris on benzoate.

    PubMed Central

    Egland, P G; Gibson, J; Harwood, C S

    1995-01-01

    The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation. PMID:7592432

  19. Ni/sup II/(dioxo(16)aneN/sub 5/)-induced methane formation from methyl coenzyme M

    SciTech Connect

    Drain, C.M.; Sable, D.B.; Corden, B.B.

    1988-07-13

    A mechanism has been previously proposed for methyl-coenzyme M (H/sub 3/CSCH/sub 2/CH/sub 2/SO/sub 3//sup /minus//) reductase where Ni/sup II/F/sub 430/ is first reduced to NiF/sub 430/, which homolytically cleaves the methyl-coenzyme M to produce methyl-Ni/sup I/F/sub 430/ followed by the protonation of methyl-Ni/sup I/F/sub 430/ to yield CH/sub 4/ and Ni/sup II/F/sub 430/. The role of the nickel ion oxidation state in methyl-coenzyme M catalysis has been examined. It was found that both the mono- and divalent oxidation states of the water soluble Ni (dioxo(16)-aneN/sub 5/), NiL, complex catalyze the methyl-coenzyme M to methane and coenzyme M. Some aqueous solutions of other nickel compounds, e.g. nickel (II) acetate, nickel(II) tetraethylenepentamine, or nickel(II) 1,4,8,11-tetraazacyclotetradecane-5,7-dione, do not convert methyl-coenzyme M to methane under argon or hydrogen. 30 references, 1 figure.

  20. Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration.

    PubMed

    Siudeja, Katarzyna; Srinivasan, Balaji; Xu, Lanjun; Rana, Anil; de Jong, Jannie; Nollen, Ellen A A; Jackowski, Suzanne; Sanford, Lynn; Hayflick, Susan; Sibon, Ody C M

    2011-12-01

    Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development. PMID:21998097

  1. Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration

    PubMed Central

    Siudeja, Katarzyna; Srinivasan, Balaji; Xu, Lanjun; Rana, Anil; de Jong, Jannie; Nollen, Ellen A A; Jackowski, Suzanne; Sanford, Lynn; Hayflick, Susan; Sibon, Ody C M

    2011-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development. PMID:21998097

  2. DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes.

    PubMed Central

    Sidhu, H; Ogden, S D; Lung, H Y; Luttge, B G; Baetz, A L; Peck, A B

    1997-01-01

    Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO2. frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a rho-independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed. PMID:9150242

  3. Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

    PubMed

    Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

    2015-06-01

    Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. PMID:25950663

  4. Role of Heme Oxygenase, Leptin, Coenzyme Q10 and Trace Elements in Pre-eclamptic Women.

    PubMed

    Abo-Elmatty, Dina M; Badawy, Ehsan A; Hussein, Jihan S; Elela, Somaya Abo; Megahed, Hoda A

    2012-10-01

    The objective of this study to evaluate heme oxygenase (COHb), leptin and coenzyme Q10 (CoQ10) in pre-eclamptic women. Also Zinc, copper, Iron, total iron binding capacity, Ferritin and uric acid were assessed. 120 female subjects were included in this study. They were divided into, 60 female with normal pregnancy attending the outpatient clinic, 60 pre-eclamptic patients were recruited from obstetrics and gynaecology department El-kasr El-Aini hospital. The results showed that in pre-eclampatic group, leptin level was significantly increased while COHb and CoQ10 was significantly decreased. It is concluded that hemeoxygenase, leptin and coenzyme CoQ10 can be considered as new markers for prediction of pre-eclampsia. PMID:24082464

  5. Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure.

    PubMed

    Desbats, Maria Andrea; Vetro, Annalisa; Limongelli, Ivan; Lunardi, Giada; Casarin, Alberto; Doimo, Mara; Spinazzi, Marco; Angelini, Corrado; Cenacchi, Giovanna; Burlina, Alberto; Rodriguez Hernandez, Maria Angeles; Chiandetti, Lino; Clementi, Maurizio; Trevisson, Eva; Navas, Placido; Zuffardi, Orsetta; Salviati, Leonardo

    2015-09-01

    Coenzyme Q10 deficiency is a clinically and genetically heterogeneous disorder, with manifestations that may range from fatal neonatal multisystem failure, to adult-onset encephalopathy. We report a patient who presented at birth with severe lactic acidosis, proteinuria, dicarboxylic aciduria, and hepatic insufficiency. She also had dilation of left ventricle on echocardiography. Her neurological condition rapidly worsened and despite aggressive care she died at 23 h of life. Muscle histology displayed lipid accumulation. Electron microscopy showed markedly swollen mitochondria with fragmented cristae. Respiratory-chain enzymatic assays showed a reduction of combined activities of complex I+III and II+III with normal activities of isolated complexes. The defect was confirmed in fibroblasts, where it could be rescued by supplementing the culture medium with 10 μM coenzyme Q10. Coenzyme Q10 levels were reduced (28% of controls) in these cells. We performed exome sequencing and focused the analysis on genes involved in coenzyme Q10 biosynthesis. The patient harbored a homozygous c.545T>G, p.(Met182Arg) alteration in COQ2, which was validated by functional complementation in yeast. In this case the biochemical and morphological features were essential to direct the genetic diagnosis. The parents had another pregnancy after the biochemical diagnosis was established, but before the identification of the genetic defect. Because of the potentially high recurrence risk, and given the importance of early CoQ10 supplementation, we decided to treat with CoQ10 the newborn child pending the results of the biochemical assays. Clinicians should consider a similar management in siblings of patients with CoQ10 deficiency without a genetic diagnosis. PMID:25564041

  6. Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

    PubMed

    Toraya, Tetsuo; Tanokuchi, Aya; Yamasaki, Ai; Nakamura, Takehiro; Ogura, Kenichi; Tobimatsu, Takamasa

    2016-01-12

    Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH. PMID:26704729

  7. The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098.

    PubMed

    Santos, Filipe; Vera, Jose L; van der Heijden, René; Valdez, Graciela; de Vos, Willem M; Sesma, Fernando; Hugenholtz, Jeroen

    2008-01-01

    The coenzyme B(12) production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B(12) gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQOP, sirA, hemACBL, and cobUSC, hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B(12) itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B(12) pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B(12) biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B(12) biosynthesis and reuterin production, which might be implicated in Lb. reuteri's success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B(12). PMID:18174128

  8. Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

    SciTech Connect

    Zhang, Jian; Fu, Yi; Li, Ge; Zhao, Richard Y.

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer Metal nanoparticle for fluorescence cell imaging. Black-Right-Pointing-Pointer Non-invasive emission detection of coenzyme in cell on time-resolved confocal microscope. Black-Right-Pointing-Pointer Near-field interaction of flavin adenine dinucleotide with silver substrate. Black-Right-Pointing-Pointer Isolation of emissions by coenzymes from cellular autofluorescence on fluorescence cell imaging. -- Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.

  9. Catalytic Formation of Hydrogen Peroxide from Coenzyme NADH and Dioxygen with a Water-Soluble Iridium Complex and a Ubiquinone Coenzyme Analogue.

    PubMed

    Suenobu, Tomoyoshi; Shibata, Satoshi; Fukuzumi, Shunichi

    2016-08-01

    A ubiquinone coenzyme analogue (Q0: 2,3-dimethoxy-5-methyl-1,4-benzoquinone) was reduced by coenzyme NADH to yield the corresponding reduced form of Q0 (Q0H2) in the presence of a catalytic amount of a [C,N] cyclometalated organoiridium complex (1: [Ir(III)(Cp*)(4-(1H-pyrazol-1-yl-κN(2))benzoic acid-κC(3))(H2O)]2SO4) in water at ambient temperature as observed in the respiratory chain complex I (Complex I). In the catalytic cycle, the reduction of 1 by NADH produces the corresponding iridium hydride complex that in turn reduces Q0 to produce Q0H2. Q0H2 reduced dioxygen to yield hydrogen peroxide (H2O2) under slightly basic conditions. Catalytic generation of H2O2 was made possible in the reaction of O2 with NADH as the functional expression of NADH oxidase in white blood cells utilizing the redox cycle of Q0 as well as 1 for the first time in a nonenzymatic homogeneous reaction system. PMID:27403568

  10. Determination of coenzyme Q10, coenzyme Q9, and melatonin contents in virgin argan oils: comparison with other edible vegetable oils.

    PubMed

    Venegas, Carmen; Cabrera-Vique, Carmen; García-Corzo, Laura; Escames, Germaine; Acuña-Castroviejo, Darío; López, Luis Carlos

    2011-11-23

    Virgin argan oil possesses high antioxidant capacity (AC), which may be partially explained by its high content in antioxidant molecules such as polyphenols and tocopherols. However, the content in other antioxidant molecules, for example, coenzyme Q10 (CoQ(10)), coenzyme Q9 (CoQ(9)), and melatonin (Mel), which have been identified in other edible vegetable oils, have not been evaluated in virgin argan oil. Consequently, it was decided to evaluate the contents of CoQ(10), CoQ(9), and Mel in virgin argan oils and compare the results to those obtained in extra virgin olive oils and some varieties of seed oils. By the use of sensitive HPLC-EC/F methods, the results showed that virgin argan oil is a rich source of CoQ(10) and Mel, but no CoQ(9) was detected. Extra virgin olive oil showed higher levels of CoQ(10) and lower levels of Mel than virgin argan oil. Between the seed oil samples, only virgin soybean oil showed higher CoQ(10) and Mel levels than virgin argan oil. The results may be relevant for the contribution of CoQ(10) and Mel to the biological activities of virgin argan oil. PMID:22007968

  11. The Phosphatase Ptc7 Induces Coenzyme Q Biosynthesis by Activating the Hydroxylase Coq7 in Yeast*

    PubMed Central

    Martín-Montalvo, Alejandro; González-Mariscal, Isabel; Pomares-Viciana, Teresa; Padilla-López, Sergio; Ballesteros, Manuel; Vazquez-Fonseca, Luis; Gandolfo, Pablo; Brautigan, David L.; Navas, Placido; Santos-Ocaña, Carlos

    2013-01-01

    The study of the components of mitochondrial metabolism has potential benefits for health span and lifespan because the maintenance of efficient mitochondrial function and antioxidant capacity is associated with improved health and survival. In yeast, mitochondrial function requires the tight control of several metabolic processes such as coenzyme Q biosynthesis, assuring an appropriate energy supply and antioxidant functions. Many mitochondrial processes are regulated by phosphorylation cycles mediated by protein kinases and phosphatases. In this study, we determined that the mitochondrial phosphatase Ptc7p, a Ser/Thr phosphatase, was required to regulate coenzyme Q6 biosynthesis, which in turn activated aerobic metabolism and enhanced oxidative stress resistance. We showed that Ptc7p phosphatase specifically activated coenzyme Q6 biosynthesis through the dephosphorylation of the demethoxy-Q6 hydroxylase Coq7p. The current findings revealed that Ptc7p is a regulator of mitochondrial metabolism that is essential to maintain proper function of the mitochondria by regulating energy metabolism and oxidative stress resistance. PMID:23940037

  12. Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans.

    PubMed

    Wei, Xuqin; Meng, Xiaolei; Chen, Yunlai; Wei, Yutuo; Du, Liqin; Huang, Ribo

    2014-01-01

    The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase. PMID:24078133

  13. An in vitro evolved glmS ribozyme has the wildtype fold but loses coenzyme dependence

    PubMed Central

    Lau, Matthew W. L.; Ferré-D’Amaré, Adrian R.

    2014-01-01

    Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, use of GlcN6P is unexpected because all other characterized self-cleaving ribozymes employ RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P, and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wildtype fold but employs divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wildtype and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme- or cation-dependent. These results indicate that a few mutations can confer novel biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist. PMID:24096303

  14. Coenzyme Q{sub 10} and alpha-tocopherol protect against amitriptyline toxicity

    SciTech Connect

    Cordero, Mario D.; Moreno-Fernandez, Ana Maria; Gomez-Skarmeta, Jose Luis; Miguel, Manuel de; Garrido-Maraver, Juan; Oropesa-Avila, Manuel; Rodriguez-Hernandez, Angeles; Navas, Placido; Sanchez-Alcazar, Jose Antonio

    2009-03-15

    Since amitriptyline is a very frequently prescribed antidepressant drug, it is not surprising that amitriptyline toxicity is relatively common. Amitriptyline toxic systemic effects include cardiovascular, autonomous nervous, and central nervous systems. To understand the mechanisms of amitriptyline toxicity we studied the cytotoxic effects of amitriptyline treatment on cultured primary human fibroblasts and zebrafish embryos, and the protective role of coenzyme Q{sub 10} and alpha-tocopherol, two membrane antioxidants. We found that amitriptyline treatment induced oxidative stress and mitochondrial dysfunction in primary human fibroblasts. Mitochondrial dysfunction in amitriptyline treatment was characterized by reduced expression levels of mitochondrial proteins and coenzyme Q{sub 10}, decreased NADH:cytochrome c reductase activity, and a drop in mitochondrial membrane potential. Moreover, and as a consequence of these toxic effects, amitriptyline treatment induced a significant increase in apoptotic cell death activating mitochondrial permeability transition. Coenzyme Q{sub 10} and alpha-tocopherol supplementation attenuated ROS production, lipid peroxidation, mitochondrial dysfunction, and cell death, suggesting that oxidative stress affecting cell membrane components is involved in amitriptyline cytotoxicity. Furthermore, amitriptyline-dependent toxicity and antioxidant protection were also evaluated in zebrafish embryos, a well established vertebrate model to study developmental toxicity. Amitriptyline significantly increased embryonic cell death and apoptosis rate, and both antioxidants provided a significant protection against amitriptyline embryotoxicity.

  15. Structure of a methyl-coenzyme M reductase from Black Sea mats that oxidize methane anaerobically.

    PubMed

    Shima, Seigo; Krueger, Martin; Weinert, Tobias; Demmer, Ulrike; Kahnt, Jörg; Thauer, Rudolf K; Ermler, Ulrich

    2012-01-01

    The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria. The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea. Here we report an X-ray structure of the 280 kDa heterohexameric ANME-1 MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate. Crystals grown from the heterogeneous sample diffract to 2.1 Å resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a F(430) modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts. PMID:22121022

  16. Coenzyme Q10 Supplementation Modulates NFκB and Nrf2 Pathways in Exercise Training

    PubMed Central

    Pala, Ragip; Orhan, Cemal; Tuzcu, Mehmet; Sahin, Nurhan; Ali, Shakir; Cinar, Vedat; Atalay, Mustafa; Sahin, Kazim

    2016-01-01

    This study reports the effects of Q10, coenzyme Q10 or ubiquinone, a component of the electron transport chain in mitochondria, on nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), inhibitors of kappa B (IκB), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and hemeoxygenase 1 (HO-1) in rats after chronic exercise training for 6 weeks. 8-week old male Wistar rats were assigned randomly to one of four treatments planned in a 2 x 2 factorial arrangement of two condition (sedentary vs. exercise training), and two coenzyme Q10 levels (0 and 300 mg/kg per day for 6 weeks). The expression levels of the target proteins were determined in the heart, liver and muscle, and biochemical parameters including creatinine, urea, glucose and lipid profile were investigated in plasma. When compared with sedentary group, significant decreases in heart, liver and muscle NFκB levels by 45%, 26% and 44% were observed in Q10 supplemented rats after exercise training, respectively, while the inhibitory protein IκB increased by 179%, 111% and 127% in heart, liver and muscle tissues. Q10 supplementation caused an increase in Nrf2 (167%, 165% and 90%) and HO-1 (107%, 156% and 114%) after exercise training in heart, liver and muscle tissues (p < 0.05). No significant change was observed in any of the parameters associated with protein, carbohydrate and lipid metabolism, except that exercise caused a decrease in plasma triglyceride, which was further decreased by Q10. In conclusion, these results suggest that Q10 modulates the expression of NFκB, IκB, Nrf2 and HO-1 in exercise training, indicating an anti-inflammatory effect of Q10 and emphasizes its role in antioxidant defense. Key points Coenzyme Q10 is a component of the electron transport chain in mitochondria which is linked to the generation of energy in the cell. Coenzyme Q10 may inhibit the peroxidation of lipids, thus acting as an antioxidant and protects tissue against oxidative injury. Using of coenzyme

  17. The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

    PubMed Central

    Liu, Yu; Jorda, Julien; Yeates, Todd O.

    2015-01-01

    ABSTRACT In Salmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology. IMPORTANCE Bacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous

  18. Beta-oxidation of very-long-chain fatty acids and their coenzyme A derivatives by human skin fibroblasts.

    PubMed

    Singh, H; Derwas, N; Poulos, A

    1987-05-01

    The beta-oxidation of lignoceric acid (C24:0), hexacosanoic acid (C26:0), and their coenzyme A derivatives was investigated in human skin fibroblast homogenates. The cofactor requirements for oxidation of lignoceric acid and hexacosanoic acid were identical but were different from their coenzyme A derivatives. For example, lignoceric acid and hexacosanoic acid oxidation was strictly ATP dependent whereas the oxidation of the corresponding coenzyme A derivatives was ATP independent. Also the rate of oxidation of coenzyme A derivatives of lignoceric acid or hexacosanoic acid was much higher compared to the free fatty acids. In patients with Zellweger's syndrome, X-linked adrenoleukodystrophy and infantile Refsum's disease, the beta-oxidation of lignoceric and hexacosanoic acids was defective whereas the oxidation of their corresponding coenzyme A derivatives was nearly normal. The results presented in this communication suggest strongly that the beta-oxidation of very-long-chain fatty acids occurs exclusively in peroxisomes. However, the coenzyme A derivatives of very-long-chain fatty acids can be oxidized in mitochondria as well as in peroxisomes. The inability of the mitochondrial system to oxidize free fatty acids may be due to its inability to convert them to their corresponding coenzyme A derivatives. Our results suggest that a specific very-long-chain fatty acyl CoA synthetase may be required for the activation of the free fatty acids and that this synthetase may be deficient in patients with Zellweger's syndrome and possibly X-linked adrenoleukodystrophy, as well. The results presented suggest that substrate specificity and the subcellular localization of the synthetase may regulate the beta-oxidation of very-long-chain fatty acids in the cell. PMID:2437859

  19. Thermodynamics of various F420 coenzyme models as sources of electrons, hydride ions, hydrogen atoms and protons in acetonitrile.

    PubMed

    Xia, Ke; Shen, Guang-Bin; Zhu, Xiao-Qing

    2015-06-14

    32 F420 coenzyme models with alkylation of the three different N atoms (N1, N3 and N10) in the core structure (XFH(-)) were designed and synthesized and the thermodynamic driving forces (defined in terms of the molar enthalpy changes or the standard redox potentials in this work) of the 32 XFH(-) releasing hydride ions, hydrogen atoms and electrons, the thermodynamic driving forces of the 32 XFH˙ releasing protons and hydrogen atoms and the thermodynamic driving forces of XF(-)˙ releasing electrons in acetonitrile were determined using titration calorimetry and electrochemical methods. The effects of the methyl group at N1, N3 and N10 and a negative charge on N1 and N10 atoms on the six thermodynamic driving forces of the F420 coenzyme models and their related reaction intermediates were examined; the results show that seating arrangements of the methyl group and the negative charge have remarkably different effects on the thermodynamic properties of the F420 coenzyme models and their related reaction intermediates. The effects of the substituents at C7 and C8 on the six thermodynamic driving forces of the F420 coenzyme models and their related reaction intermediates were also examined; the results show that the substituents at C7 and C8 have good Hammett linear free energy relationships with the six thermodynamic parameters. Meanwhile, a reasonable determination of possible reactions between members of the F420 family and NADH family in vivo was given according to a thermodynamic analysis platform constructed using the elementary step thermodynamic parameter of F420 coenzyme model 2FH(-) and NADH model MNAH releasing hydride ions in acetonitrile. The information disclosed in this work can not only fill a gap in the chemical thermodynamics of F420 coenzyme models as a class of very important organic sources of electrons, hydride ions, hydrogen atoms and protons, but also strongly promote the fast development of the chemistry and applications of F420 coenzyme

  20. Spectroscopic and computational studies of reduction of the metalversus the tetrapyrrole ring of coenzyme F-430 from methyl-coenzyme Mreductase

    SciTech Connect

    Dey, Mishtu; Kunz, Ryan C.; van Heuvelen, Katherine M.; Craft,Jennifer L.; Horng, Yih-Chern; Tang, Qun; Bocian, David F.; George, SimonJ.; Brunold, Thomas C.; Ragsdale, Stephen W.

    2006-06-30

    Methyl-coenzyme M reductase (MCR) catalyzes the final stepin methane biosynthesis by methanogenic archaea and contains aredox-active nickel tetrahydrocorphin, coenzyme F430, at its active site.Spectroscopic and computational methods have been used to study a novelform of the coenzyme, called F330, which is obtained by reducing F430with sodium borohydride (NaBH4). F330 exhibits a prominent absorptionpeak at 330 nm, which is blue shifted by 100 nm relative to F430. Massspectrometric studies demonstrate that the tetrapyrrole ring in F330 hasundergone reduction, on the basis of the incorporation of protium (ordeuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- andtwo-dimensional NMR studies show that the site of reduction is theexocyclic ketone group of the tetrahydrocorphin. Resonance Raman studiesindicate that elimination of this pibond increases the overall pi-bondorder in the conjugative framework. X-ray absorption, magnetic circulardichroism, and computational results show that F330 contains low-spinNi(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ringbut not the metal. Conversely, reduction of F430 with Ti(III) citrate togenerate F380 (corresponding to the active MCRred1 state) reduces theNi(II) to Ni(I) but does not reduce the tetrapyrrole ring system, whichis consistent with other studies [Piskorski, R., and Jaun, B. (2003) J.Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol.Inorg. Chem. 9, 77-89]. The distinct origins of the absorption bandshifts associated with the formation of F330 and F380 are discussedwithin the framework of our computational results. These studies on thenature of the product(s) of reduction of F430 are of interest in thecontext of the mechanism of methane formation by MCR and in relation tothe chemistry of hydroporphinoid systems in general. The spectroscopicand time-dependent DFT calculations add important insight into theelectronic structure of the nickel hydrocorphinate in its Ni(II) and

  1. Monolignol Pathway 4-Coumaric Acid:Coenzyme A Ligases in Populus. trichocarpa: Novel Specificity, Metabolic Regulation, and Simulation of Coenzyme A Ligation Fluxes1[W

    PubMed Central

    Chen, Hsi-Chuan; Song, Jina; Williams, Cranos M.; Shuford, Christopher M.; Liu, Jie; Wang, Jack P.; Li, Quanzi; Shi, Rui; Gokce, Emine; Ducoste, Joel; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.

    2013-01-01

    4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stem-differentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis. PMID:23344904

  2. The use of coenzyme Q0 as a template in the development of a molecularly imprinted polymer for the selective recognition of coenzyme Q10.

    PubMed

    Contin, Mario; Flor, Sabrina; Martinefski, Manuela; Lucangioli, Silvia; Tripodi, Valeria

    2014-01-01

    In this work, a novel molecularly imprinted polymer (MIP) for use as a solid phase extraction sorbent was developed for the determination of coenzyme Q10 (CoQ10) in liver extract. CoQ10 is an essential cofactor in mitochondrial oxidative phosphorylation and a powerful antioxidant agent found in low concentrations in biological samples. This fact and its high hydrophobicity make the analysis of CoQ10 technically challenging. Accordingly, a MIP was synthesised using coenzyme Q0 as the template, methacrylic acid as the functional monomer, acetonitrile as the porogen, ethylene glycol dimethacrylate as the crosslinker and benzoyl peroxide as the initiator. Various parameters affecting the polymer preparation and extraction efficiency were evaluated. Morphological characterisation of the MIP and its proper comparison with C18 as a sorbent in solid phase extraction were performed. The optimal conditions for the molecularly imprinted solid phase extraction (MISPE) consisted of 400 μL of sample mixed with 30 mg of MIP and 600 μL of water to reach the optimum solution loading. The loading was followed by a washing step consisting of 1 mL of a 1-propanol solution (1-propanol:water, 30:70,v/v) and elution with 1 mL of 1-propanol. After clean-up, the CoQ10 in the samples was analysed by high performance liquid chromatography. The extraction recoveries were higher than 73.7% with good precision (3.6-8.3%). The limits of detection and quantification were 2.4 and 7.5 μg g(-1), respectively, and a linear range between 7.5 and 150 μg g(-1) of tissue was achieved. The new MISPE procedure provided a successful clean-up for the determination of CoQ10 in a complex matrix. PMID:24356222

  3. Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Steindorf, Astrid; Alber, Birgit E; Fuchs, Georg

    2006-04-01

    The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle. PMID:16547052

  4. Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

    SciTech Connect

    Jomrit, Juntratip; Summpunn, Pijug; Meevootisom, Vithaya; Wiyakrutta, Suthep

    2011-02-25

    Research highlights: {yields} Stereochemical mechanism of PLP enzymes is important but difficult to determine. {yields} This new method is significantly less complicated than the previous ones. {yields} This assay is as sensitive as the radioactive based method. {yields} LC-MS/MS positively identify the analyte coenzyme. {yields} The method can be used with enzyme whose apo form is unstable. -- Abstract: A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in {sup 2}H{sub 2}O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-{sup 2}H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The {sup 2}H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the {sup 2}H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of {sup 2}H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.

  5. A new role for coenzyme F420 in aflatoxin reduction by soil mycobacteria.

    PubMed

    Graham, David E

    2010-11-01

    Hepatotoxic aflatoxins have found a worthy adversary in two new families of bacterial oxidoreductases. These enzymes use the reduced coenzyme F420 to initiate the degradation of furanocoumarin compounds, including the major mycotoxin products of Aspergillus flavus. Along with pyridoxal 5'-phosphate synthases and aryl nitroreductases, these proteins form a large and versatile superfamily of flavin and deazaflavin-dependent oxidoreductases. F420-dependent members of this family appear to share a common mechanism of hydride transfer from the reduced, low-potential deazaflavin to the electron-deficient ring systems of their substrates. PMID:21038477

  6. Regeneration of Nicotinamide Coenzymes: Principles and Applications for the Synthesis of Chiral Compounds

    NASA Astrophysics Data System (ADS)

    Weckbecker, Andrea; Gröger, Harald; Hummel, Werner

    Dehydrogenases which depend on nicotinamide coenzymes are of increasing interest for the preparation of chiral compounds, either by reduction of a prochiral precursor or by oxidative resolution of their racemate. The regeneration of oxidized and reduced nicotinamide cofactors is a very crucial step because the use of these cofactors in stoichiometric amounts is too expensive for application. There are several possibilities to regenerate nicotinamide cofactors: established methods such as formate/formate dehydrogenase (FDH) for the regeneration of NADH, recently developed electrochemical methods based on new mediator structures, or the application of gene cloning methods for the construction of "designed" cells by heterologous expression of appropriate genes.

  7. MicroCommentary: A New Role for Coenzyme F420 in Aflatoxin Reduction by Soil Mycobacteria

    SciTech Connect

    Graham, David E

    2010-01-01

    Hepatotoxic aflatoxins have found a worthy adversary in two new families of bacterial oxidoreductases. These enzymes use the reduced coenzyme F420 to initiate the degradation of furanocoumarin compounds, including the major mycotoxin products of Aspergillus flavus. Along with pyridoxalamine 5 -phosphate oxidases and aryl nitroreductases, these proteins form a large and versatile superfamily of flavin and deazaflavin-dependent oxidoreductases. F420-dependent members of this family appear to share a common mechanism of hydride transfer from the reduced deazaflavin to the electron-deficient ring systems of their substrates.

  8. Site-Selective Synthesis of (15)N- and (13)C-Enriched Flavin Mononucleotide Coenzyme Isotopologues.

    PubMed

    Neti, Syam Sundar; Poulter, C Dale

    2016-06-17

    Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with (15)N and (13)C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis. PMID:27176708

  9. Structure of the corrinoid:coenzyme M methyltransferase MtaA from Methanosarcina mazei.

    PubMed

    Hoeppner, Astrid; Thomas, Frank; Rueppel, Alma; Hensel, Reinhard; Blankenfeldt, Wulf; Bayer, Peter; Faust, Annette

    2012-11-01

    The zinc-containing corrinoid:coenzyme M methyltransferase MtaA is part of the methanol-coenzyme M-methyltransferase complex of Methanosarcina mazei. The whole complex consists of three subunits: MtaA, MtaB and MtaC. The MtaB-MtaC complex catalyses the cleavage of methanol (bound to MtaB) and the transfer of the methyl group onto the cobalt of cob(I)alamin (bound to MtaC). The MtaA-MtaC complex catalyses methyl transfer from methyl-cob(III)alamin (bound to MtaC) to coenzyme M (bound to MtaA). The crystal structure of the MtaB-MtaC complex from M. barkeri has previously been determined. Here, the crystal structures of MtaA from M. mazei in a substrate-free but Zn(2+)-bound state and in complex with Zn(2+) and coenzyme M (HS-CoM) are reported at resolutions of 1.8 and 2.1 Å, respectively. A search for homologous proteins revealed that MtaA exhibits 23% sequence identity to human uroporphyrinogen III decarboxylase, which has also the highest structural similarity (r.m.s.d. of 2.03 Å for 306 aligned amino acids). The main structural feature of MtaA is a TIM-barrel-like fold, which is also found in all other zinc enzymes that catalyse thiol-group alkylation. The active site of MtaA is situated at the narrow bottom of a funnel such that the thiolate group of HS-CoM points towards the Zn(2+) ion. The Zn(2+) ion in the active site of MtaA is coordinated tetrahedrally via His240, Cys242 and Cys319. In the substrate-free form the fourth ligand is Glu263. Binding of HS-CoM leads to exchange of the O-ligand of Glu263 for the S-ligand of HS-CoM with inversion of the zinc geometry. The interface between MtaA and MtaC for transfer of the methyl group from MtaC-bound methylcobalamin is most likely to be formed by the core complex of MtaB-MtaC and the N-terminal segment (a long loop containing three α-helices and a β-hairpin) of MtaA, which is not part of the TIM-barrel core structure of MtaA. PMID:23090404

  10. 3-hydroxy 3-methylglutaryl coenzyme A reductase: a new biomarker of fish exposure to water pollution.

    PubMed

    Pallottini, Valentina; Scalici, Massimiliano; Gibertini, Giancarlo; Marino, Maria; Trentalance, Anna

    2010-10-01

    The aim of this study was to identify a new putative biomarker in Salmo trutta exposed to water pollution. Variations in the levels of hepatic 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMG-CoAR), the rate-limiting enzyme of cholesterol biosynthesis, were compared to heat shock protein 70 and hypoxia inducible factor α, biomarkers of pollution exposure and lowered O₂, respectively. The results confirm that HMG-CoAR levels increase in polluted water irrespective of water temperature or O₂ content, indicating that HMG-CoAR could be used as a specific biomarker for water pollution. PMID:20835703

  11. An Improvement of Oxidative Stress in Diabetic Rats by Ubiquinone-10 and Ubiquinol-10 and Bioavailability after Short- and Long-Term Coenzyme Q10 Supplementation.

    PubMed

    Prangthip, Pattaneeya; Kettawan, Aikkarach; Posuwan, Juthathip; Okuno, Masaaki; Okamoto, Tadashi

    2016-11-01

    This study explored effects of ubiquinol-10 and ubiquinone-10, two different forms of coenzyme Q10, in diabetic rats. Oxidative stress is characterized by the depletion of antioxidant defenses and overproduction of free radicals that might contribute to, and even accelerate, the development of diabetes mellitus (DM) complications. Coenzyme Q10 was administered orally to diabetic rats and oxidative stress markers were then assessed. Bioavailability in normal rats was additionally assessed in various tissues and subcellular fractions after short-term and long-term coenzyme Q10 supplementation. Elevated nonfasting blood glucose and blood pressure in diabetic rats were decreased by ubiquinone-10. Both ubiquinol-10 and ubiquinone-10 ameliorated oxidative stress, based on assays for reactive oxygen metabolites and malondialdehyde. Coenzyme Q10 levels increased with both treatments and liver nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme Q reductase with ubiquinone-10. Ubiquinol-10 was better absorbed in the liver and pancreas than ubiquinone-10, though both were similarly effective. In bioavailability study, a longer period of coenzyme Q10 supplementation did not lead to its accumulation in tissues or organelles. Both forms of coenzyme Q10 reduced oxidative stress in diabetic rats. Long-term supplementation of coenzyme Q10 appeared to be safe. PMID:27064932

  12. Effects of menopause and hormone replacement therapy on serum levels of coenzyme Q10 and other lipid-soluble antioxidants.

    PubMed

    Palan, Prabhudas R; Connell, Kathleen; Ramirez, Elizabeth; Inegbenijie, Christian; Gavara, Rachana Y; Ouseph, Jacob A; Mikhail, Magdy S

    2005-01-01

    The present study examines the influence of menopause and hormone replacement therapy (HRT) on serum levels of coenzyme Q(10) and other lipid-soluble antioxidants in normal women. Serum levels of coenzyme Q(10), alpha-tocopherol, gamma-tocopherol, beta-carotene and lycopene in 50 premenopausal women (not using oral contraceptives), 33 healthy postmenopausal and 15 postmenopausal women on HRT ("Prempo"; combination of 0.625 mg conjugated estrogen and 2.5 mg medroxyprogesterone acetate) were measured by high-pressure liquid chromatography. Lipid profiles were also analyzed. Significantly higher serum coenzyme Q(10) and alpha-tocopherol levels were detected in postmenopausal compared with premenopausal women (P < 0.05, and < 0.001); whereas, in postmenopausal subjects on HRT, we detected a significant decrease in coenzyme Q(10) and gamma-tocopherol levels (P < 0.001, and < 0.05) and increased alpha-tocopherol levels (P < 0.05). Serum levels of beta-carotene, lycopene, LDL, HDL, cholesterol and triglyceride were comparable among the study groups. Coenzyme Q(10) is postulated to be involved in preventing cardiovascular disease (CVD) because of its bioenergetics role in the mitochondrial respiratory chain and its antioxidant properties at the mitochondrial and extramitochondrial levels. The decrease in serum concentrations of coenzyme Q(10), produced by HRT, may promote oxygen free radical-induced membrane damage and may, thus alter cardiovascular risk in postmenopausal women. HRT-induced reductions in lipid-soluble antioxidant(s) levels, and its potential consequences on CVD, needs to be further investigated. PMID:16873930

  13. Structural Analysis of a Ni-Methyl Species in Methyl-Coenzyme M Reductase from Methanothermobacter marburgensis

    SciTech Connect

    Cedervall, Peder E.; Dey, Mishtu; Li, Xianghui; Sarangi, Ritimukta; Hedman, Britt; Ragsdale, Stephen W.; Wilmot, Carrie M.

    2012-02-15

    We present the 1.2 {angstrom} resolution X-ray crystal structure of a Ni-methyl species that is a proposed catalytic intermediate in methyl-coenzyme M reductase (MCR), the enzyme that catalyzes the biological formation of methane. The methyl group is situated 2.1 {angstrom} proximal of the Ni atom of the MCR coenzyme F{sub 430}. A rearrangement of the substrate channel has been posited to bring together substrate species, but Ni(III)-methyl formation alone does not lead to any observable structural changes in the channel.

  14. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum

    PubMed Central

    Huang, Shi-Ping; Cheng, Hong-Mei; Wang, Peng; Zhu, Guo-Ping

    2016-01-01

    Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP+-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP+ over NAD+ in the presence of Mg2+ and Mn2+, respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn2+) and 65 °C (with Mg2+), and pH 7.5 (with Mn2+) and pH 8.0 (with Mg2+). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn2+ or Mg2+. Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP+ use by the IDH family. PMID:26927087

  15. Inhibition of acetyl-coenzyme A carboxylase by two classes of grass-selective herbicides

    SciTech Connect

    Rendina, A.R.; Craig-Kennard, A.C.; Beaudoin, J.D.; Breen, M.K. )

    1990-05-01

    The selective grass herbicides diclofop, haloxyfop, and trifop (((aryloxy)phenoxy)propionic acids) and alloxydim, sethoxydim, and clethodim (cyclohexanediones) are potent, reversible inhibitors of acetyl-coenzyme A carboxylase (ACC) partially purified from barley, corn, and wheat. Although inhibition of the wheat enzyme by clethodim and diclofop is noncompetitive versus each of the substrates adenosine triphosphate (ATP), HCO{sub 3}{sup {minus}}, and acetyl-coenzyme A (acetyl-CoA), diclofop and clethodim are nearly competitive versus acetyl-CoA since the level of inhibition is most sensitive to the concentration of acetyl-CoA (K{sub is} < K{sub ii}). To conclusively show whether the herbicides interact at the biotin carboxylation site or the carboxyl transfer site, the inhibition of isotope exchange and partial reactions catalyzed at each site was studied with the wheat enzyme. Only the ({sup 14}C)acetyl-CoA-malonyl-CoA exchange and decarboxylation of ({sup 14}C)malonyl-CoA reactions are strongly inhibited by clethodim and diclofop, suggesting that the herbicides interfere with the carboxyl transfer site rather than the biotin carboxylation site of the enzyme. Double-inhibition studies with diclofop and clethodim suggest that the ((aryloxy)phenoxy)propionic acid and cyclohexanedione herbicides may bind to the same region of the enzyme.

  16. Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis.

    PubMed

    Stefely, Jonathan A; Reidenbach, Andrew G; Ulbrich, Arne; Oruganty, Krishnadev; Floyd, Brendan J; Jochem, Adam; Saunders, Jaclyn M; Johnson, Isabel E; Minogue, Catherine E; Wrobel, Russell L; Barber, Grant E; Lee, David; Li, Sheng; Kannan, Natarajan; Coon, Joshua J; Bingman, Craig A; Pagliarini, David J

    2015-01-01

    The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways. PMID:25498144

  17. Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

    PubMed Central

    Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne; Oruganty, Krishnadev; Floyd, Brendan J.; Jochem, Adam; Saunders, Jaclyn M.; Johnson, Isabel E.; Minogue, Catherine E.; Wrobel, Russell L.; Barber, Grant E.; Lee, David; Li, Sheng; Kannan, Natarajan; Coon, Joshua J.; Bingman, Craig A.; Pagliarini, David J.

    2014-01-01

    SUMMARY The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation, but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways. PMID:25498144

  18. Reversal of statin-induced memory dysfunction by co-enzyme Q10: a case report.

    PubMed

    Okeahialam, Basil N

    2015-01-01

    Statins are useful in the armamentarium of the clinician dealing with dyslipidemia, which increases cardiovascular morbi-mortality in hypertensive and diabetic patients among others. Dyslipidemia commonly exists as a comorbidity factor in the development of atherosclerotic cardiovascular disease. Use of statins is however associated with side effects which at times are so disabling as to interfere with activities of daily living. There are various ways of dealing with this, including use of more water-soluble varieties, intermittent dosing, or use of statin alternatives. Of late, use of co-enzyme Q10 has become acceptable for the muscle side effects. Only one report of any benefit on the rarely reported memory side effect was encountered by the author in the search of English medical literature. This is a report of a documented case of a Nigerian woman with history of statin intolerance in this case, memory dysfunction despite persisting dyslipidemia comorbidity. Her memory dysfunction side effect which interfered with activities of daily living and background muscle pain cleared when coenzyme Q10 was administered alongside low dose statin. Her lipid profile normalized and has remained normal. It is being recommended for use when statin side effects (muscle- and memory-related) impair quality of life and leave patient at dyslipidemia-induced cardiovascular morbi-mortality. PMID:26604775

  19. Effect of Coenzyme Q on Serum Levels of Creatine Phosphokinase in Preclinical Muscular Dystrophy*†

    PubMed Central

    Folkers, Karl; Nakamura, Ryo; Littarru, Gian Paolo; Zellweger, Hans; Brunkhorst, John B.; Williams, Coyle W.; Langston, John H.

    1974-01-01

    Coenzyme Q10 (CoQ10) exists in human tissue, and is indispensable to mitochondrial enzymes of respiration. CoQ was administered to children with preclinical muscular dystrophy, CoQ enzymology was emphasized, and serum creatine phosphokinase, CPK, (ATP:creatine N-phosphotransferase, EC 2.7.3.2) was repeatedly monitored. A 40-week treatment of an infant, 1-2 years of age, reduced serum CPK (P < 0.001; total CPK assays, 76). A 40-week treatment of a boy, 3-5 years of age, reduced serum CPK (P < 0.01); treatment through 80 weeks reduced CPK (P < 0.001; total CPK assays, 118). This response of preclinical dystrophy to CoQ implies a deficiency of CoQ in skeletal muscle that was actually found previously by assay of the activity of the succinate dehydrogenase:coenzyme Q10 reductase of the rectus abdominis. The relationships among a CoQ deficiency in muscle, serum CPK, and use of CPK in muscle are uncertain; however, restoration of CoQ enzyme activity in muscle by oral administration of CoQ could lead to increased use of CPK in muscle to form phosphocreatine from creatine and ATP, with a corresponding decrease in serum levels of CPK. The great excess of CPK in serum comes from deteriorating muscle in which CPK is below normal. PMID:4525474

  20. New insights into the chemistry of Coenzyme Q-0: A voltammetric and spectroscopic study.

    PubMed

    Gulaboski, Rubin; Bogeski, Ivan; Kokoskarova, Pavlinka; Haeri, Haleh H; Mitrev, Sasa; Stefova, Marina; Stanoeva, Jasmina Petreska; Markovski, Velo; Mirčeski, Valentin; Hoth, Markus; Kappl, Reinhard

    2016-10-01

    Coenzyme Q-0 (CoQ-0) is the only Coenzyme Q lacking an isoprenoid group on the quinoid ring, a feature important for its physico-chemical properties. Here, the redox behavior of CoQ-0 in buffered and non-buffered aqueous media was examined. In buffered aqueous media CoQ-0 redox chemistry can be described by a 2-electron-2-proton redox scheme, characteristic for all benzoquinones. In non-buffered media the number of electrons involved in the electrode reaction of CoQ-0 is still 2; however, the number of protons involved varies between 0 and 2. This results in two additional voltammetric signals, attributed to 2-electrons-1H(+) and 2-electrons-0H(+) redox processes, in which mono- and di-anionic compounds of CoQ-0 are formed. In addition, CoQ-0 exhibits a complex chemistry in strong alkaline environment. The reaction of CoQ-0 and OH(-) anions generates several hydroxyl derivatives as products. Their structures were identified with HPLC/MS. The prevailing radical reaction mechanism was analyzed by electron paramagnetic resonance spectroscopy. The hydroxyl derivatives of CoQ-0 have a strong antioxidative potential and form stable complexes with Ca(2+) ions. In summary, our results allow mechanistic insights into the redox properties of CoQ-0 and its hydroxylated derivatives and provide hints on possible applications. PMID:27268099

  1. Molecular characterization of methanogenic N(5)-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase.

    PubMed

    Upadhyay, Vikrant; Ceh, Katharina; Tumulka, Franz; Abele, Rupert; Hoffmann, Jan; Langer, Julian; Shima, Seigo; Ermler, Ulrich

    2016-09-01

    Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS. PMID:27342374

  2. Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects

    PubMed Central

    Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

    2015-01-01

    Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5′-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. PMID:25950663

  3. Role of active site loop in coenzyme binding and flavin reduction in cytochrome P450 reductase.

    PubMed

    Mothersole, Robert G; Meints, Carla E; Louder, Alex; Wolthers, Kirsten R

    2016-09-15

    Cytochrome P450 reductase (CPR) contains a loop within the active site (comprising Asp(634), Ala(635), Arg(636) and Asn(637); human CPR numbering) that relocates upon NADPH binding. Repositioning of the loop triggers the reorientation of an FAD-shielding tryptophan (Trp(679)) to a partially stacked conformer, reducing the energy barrier for displacement of the residue by the NADPH nicotinamide ring: an essential step for hydride transfer. We used site-directed mutagenesis and kinetic analysis to investigate if the amino acid composition of the loop influences the catalytic properties of CPR. The D634A and D634N variants elicited a modest increase in coenzyme binding affinity coupled with a 36- and 10-fold reduction in cytochrome c(3+) turnover and a 17- and 3-fold decrease in the pre-steady state rate of flavin reduction. These results, in combination with a reduction in the kinetic isotope effect for hydride transfer, suggest that diminished activity is due to destabilization of the partially stacked conformer of Trp(677) and slower release of NADP(+). In contrast, R636A, R636S and an A635G/R636S double mutant led to a modest increase in cytochrome c(3+) reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer. A potential mechanism by which Arg(636) influences catalysis is discussed. PMID:27461959

  4. 3-Hydroxybenzoate:coenzyme A ligase from cell cultures of Centaurium erythraea: isolation and characterization.

    PubMed

    Barillas, W; Beerhues, L

    2000-02-01

    In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a seven-step-procedure. The enzyme was an AMP-forming CoA ligase with a Km = 14.7 microM for 3-hydroxybenzoic acid, 8.5 microM for coenzyme A and 229 microM for ATP. The pH and temperature optima were 7.5 and 35 degrees C, respectively. In SDS-PAGE, two polypeptides of Mr 41,500 and 40,500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their N-termini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation. 3HBL had a native Mr of approx. 50,000 and is thus active as a monomer. PMID:10746747

  5. [Cloning and tissue expression of 4-coumarate coenzyme A ligase gene in Angelica sinensis].

    PubMed

    Wen, Sui-chao; Wang, Yin-quan; Luo, Jun; Xia, Qi; Fan, Qin; Li, Shu-nan; Wang, Zhen-heng

    2015-12-01

    4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis. PMID:27245029

  6. In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold.

    PubMed

    Lau, Matthew W L; Ferré-D'Amaré, Adrian R

    2016-08-15

    Uniquely among known natural ribozymes that cleave RNA sequence-specifically, the glmS ribozyme-riboswitch employs a small molecule, glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. In vitro selection was employed to search for coenzyme-independent variants of this ribozyme. In addition to shedding light on the catalytic mechanism of the ribozyme, such variants could resemble the evolutionary ancestors of the modern, GlcN6P-regulated ribozyme-riboswitch. A mutant pool was constructed such that the secondary structure elements, which define the triply-pseudoknotted global fold of the ribozyme, was preserved. A stringent selection scheme that relies on thiol-mercury affinity chromatography for separating active and inactive sequences ultimately yielded a triple mutant with a cleavage rate exceeding 3min(-1) that only requires divalent cations for activity. Mutational analysis demonstrated that a point reversion of the variant toward the wild-type sequence was sufficient to partially restore GlcN6P-dependence, suggesting that coenzyme dependence can be readily be acquired by RNAs that adopt the glmS ribozyme fold. The methods employed to perform this selection experiment are described in detail in this review. PMID:27130889

  7. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum.

    PubMed

    Huang, Shi-Ping; Cheng, Hong-Mei; Wang, Peng; Zhu, Guo-Ping

    2016-01-01

    Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family. PMID:26927087

  8. High-resolution neutron crystallographic studies of the hydration of the coenzyme cob(II)alamin

    SciTech Connect

    Jogl, Gerwald; Wang, Xiaoping; Mason, Sax A.; Kovalevsky, Andrey; Mustyakimov, Marat; Fisher, Zöe; Hoffman, Christina; Kratky, Christoph; Langan, Paul

    2011-06-01

    High-resolution crystallographic studies of the hydration of the coenzyme cob(II)alamin have provided hydrogen-bond parameters of unprecedented accuracy for a biomacromolecule. The hydration of the coenzyme cob(II)alamin has been studied using high-resolution monochromatic neutron crystallographic data collected at room temperature to a resolution of 0.92 Å on the original D19 diffractometer with a prototype 4° × 64° detector at the high-flux reactor neutron source run by the Institute Laue–Langevin. The resulting structure provides hydrogen-bonding parameters for the hydration of biomacromolecules to unprecedented accuracy. These experimental parameters will be used to define more accurate force fields for biomacromolecular structure refinement. The presence of a hydrophobic bowl motif surrounded by flexible side chains with terminal functional groups may be significant for the efficient scavenging of ligands. The feasibility of extending the resolution of this structure to ultrahigh resolution was investigated by collecting time-of-flight neutron crystallographic data during commissioning of the TOPAZ diffractometer with a prototype array of 14 modular 2° × 21° detectors at the Spallation Neutron Source run by Oak Ridge National Laboratory.

  9. Plasma coenzyme Q10 levels in type 2 diabetic patients with retinopathy

    PubMed Central

    Ates, Orhan; Bilen, Habip; Keles, Sadullah; Alp, H. Hakan; Keleş, Mevlüt Sait; Yıldırım, Kenan; Öndaş, Osman; Pınar, L. Can; Civelekler, Mustafa; Baykal, Orhan

    2013-01-01

    AIM To determine the relationship between proliferative diabetic retinopathy (PDRP) and plasma coenzyme Q10(CoQ10) concentration. METHODS Patients with type 2 diabetes and PDRP were determined to be the case group (n=50). The control group was consist of healthy individuals (n=50). Plasma CoQ10 and malondialdehyde (MDA) levels were measured in both groups. RESULTS Ubiquinone-10 (Coenzyme Q10) levels in PDRP and control subjects are 3.81±1.19µmol/L and 1.91±0.62µmol/L, respectively. Plasma MDA levels in PDRP and control subjects were 8.16±2µmol/L and 3.44±2.08µmol/L, respectively. Ratio of Ubiquinol-10/ubiquinone-10 in PDRP and control subjects were 0.26±0.16 and 1.41±0.68, respectively. CONCLUSION The ratio of ubiquinol-10/ubiquinone-10 is found lower in patients with PDRP. High levels of plasma ubiquinol-10/ubiquinone-10 ratio indicate the protective effect on diabetic retinopathy. PMID:24195048

  10. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  11. Assay for methylmalonyl coenzyme A mutase activity based on determination of succinyl coenzyme A by ultrahigh-performance liquid chromatography tandem mass spectrometry.

    PubMed

    Gotoh, Kana; Nakajima, Yoko; Tajima, Go; Hotta, Yuji; Kataoka, Tomoya; Kawade, Yoshihiro; Sugiyama, Naruji; Ito, Tetsuya; Kimura, Kazunori; Maeda, Yasuhiro

    2015-07-01

    Methylmalonic acidemia (MMA) is an inherited metabolic disease. In this condition, metabolism from methylmalonyl coenzyme A (CoA) to succinyl-CoA is inhibited because of either low methylmalonyl-CoA mutase (MCM) activity or adenosylcobalamin deficiency owing to altered vitamin B12 metabolism. A high-precision assay for detecting MCM activity would facilitate not only MMA diagnosis but also the ability to determine the severity of MMA. We developed an MCM assay method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) that involves the determination of succinyl-CoA, which is formed in an enzyme reaction, using peripheral lymphocytes. Using 0.05, 0.5, and 5 μmol/L succinyl-CoA, the intra-assay coefficient of variation (CV) was less than 5.2% and the inter-assay CV was less than 8.7%. The MCM activities of five healthy individuals and four patients were investigated with this assay. The MCM activities of the patients were very low in relation to those of healthy individuals. Together, these results show that the UPLC-MS/MS method is useful for a detailed MCM activity assay. PMID:26018627

  12. [Corrective effect of trimethylglycine on the nicotinamide coenzyme and adenine nucleotide content of the tissues in experimental atherosclerosis].

    PubMed

    Zapadniuk, V I; Chekman, I S; Panteleĭmonova, T N; Tumanov, V A

    1986-01-01

    Experiments on adult rabbits with experimental atherosclerosis induced by cholesterol (0.25 g/kg for 90 days) showed that chronic administration of trimethylglycine (1.5 g/kg for 30 days) prevented a decrease of the liver and myocardium content of nicotinamide coenzymes and adenine nucleotides. PMID:3758334

  13. Regulation of 4CL, encoding 4-coumarate: coenzyme A ligase, expression in kenaf under diverse stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We cloned the full length 4CL ortholog encoding 4-coumarate: coenzymeA ligase from kenaf (Hibiscus cannabiuns) using degenerate primers and RACE (rapid amplification of cDNA ends) systems. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic ac...

  14. Absence of malonyl coenzyme A decarboxylase in mice increases cardiac glucose oxidation and protects the heart from ischemic injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acute pharmacological inhibition of cardiac malonyl coenzyme A decarboxylase (MCD) protects the heart from ischemic damage by inhibiting fatty acid oxidation and stimulating glucose oxidation. However, it is unknown whether chronic inhibition of MCD results in altered cardiac function, energy metabo...

  15. Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid1[W

    PubMed Central

    Sonnante, Gabriella; D'Amore, Rosalinda; Blanco, Emanuela; Pierri, Ciro L.; De Palma, Monica; Luo, Jie; Tucci, Marina; Martin, Cathie

    2010-01-01

    Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes. PMID:20431089

  16. The Impact of Coenzyme Q[subscript10] Supplement on the Indicators of Muscle Damage in Young Male Skiing Athletes

    ERIC Educational Resources Information Center

    Demirci, Nevzat

    2015-01-01

    This study was conducted in order to know the impact of coenzyme Q[subscript 10] (CoQ[subscript 10]) supplement on the muscle damage and total oxidant (TOS) enzyme levels of young skiing athletes during exercise. 15 male athletes were used for two weeks in the study. The athletes were divided into three groups: the control group and two subject…

  17. Structural determinants in bacterial 2-keto-3-deoxy-D-gluconate dehydrogenase KduD for dual-coenzyme specificity.

    PubMed

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

    2016-07-01

    Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/β/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc. PMID:27028675

  18. The STM4195 Gene Product (PanS) Transports Coenzyme A Precursors in Salmonella enterica

    PubMed Central

    Ernst, Dustin C.

    2015-01-01

    ABSTRACT Coenzyme A (CoA) is a ubiquitous coenzyme involved in fundamental metabolic processes. CoA is synthesized from pantothenic acid by a pathway that is largely conserved among bacteria and eukaryotes and consists of five enzymatic steps. While higher organisms, including humans, must scavenge pantothenate from the environment, most bacteria and plants are capable of de novo pantothenate biosynthesis. In Salmonella enterica, precursors to pantothenate can be salvaged, but subsequent intermediates are not transported due to their phosphorylated state, and thus the pathway from pantothenate to CoA is considered essential. Genetic analyses identified the STM4195 gene product of Salmonella enterica serovar Typhimurium as a transporter of pantothenate precursors, ketopantoate and pantoate and, to a lesser extent, pantothenate. Further results indicated that STM4195 transports a product of CoA degradation that serves as a precursor to CoA and enters the biosynthetic pathway between PanC and CoaBC (dfp). The relevant CoA derivative is distinguishable from pantothenate, pantetheine, and pantethine and has spectral properties indicating the adenine moiety of CoA is intact. Taken together, the results presented here provide evidence of a transport mechanism for the uptake of ketopantoate, pantoate, and pantothenate and demonstrate a role for STM4195 in the salvage of a CoA derivative of unknown structure. The STM4195 gene is renamed panS to reflect participation in pantothenate salvage that was uncovered herein. IMPORTANCE This manuscript describes a transporter for two pantothenate precursors in addition to the existence and transport of a salvageable coenzyme A (CoA) derivative. Specifically, these studies defined a function for an STM protein in S. enterica that was distinct from the annotated role and led to its designation as PanS (pantothenate salvage). The presence of a salvageable CoA derivative and a transporter for it suggests the possibility that this

  19. Ultra-fast simultaneous detection of obesity-related coenzymes in mice using microchip electrophoresis with a LIF detector.

    PubMed

    Lee, Hee Gu; Kumar, K S; Soh, Ju-Ryoun; Cha, Youn-Soo; Kang, Seong Ho

    2008-06-30

    Hepatic acyl-coenzyme A synthetase (ACS), carnitine palmitoyltransferase-I (CPT-I) and acetyl coenzyme A carboxylase (ACC) are coenzymes associated with the genetic type of obesity in animal models. This paper reports the use of microchip electrophoresis (ME) with a laser-induced fluorescence (LIF) detector based on a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the amplified DNA fragments of these coenzymes (ACS, CPT-I and ACC) in the mRNA extracted from mice. DNA fragments ranging from 50 to 2652 bp were well resolved using this procedure with a running buffer (1x TBE), 0.5% polyvinylpyrrolidone (M(r) 1,000,000) as the coating gel and 0.7% polyethyleneoxide (M(r) 8,000,000) as the sieving gel at pH 8.30. The separation of the three RT-PCR products was achieved by ME in a single-run within 17 s using programmed field strength gradients (PFSG) (470 V cm(-1) for 9 s, 205.8 V cm(-1) for 2 s, 411.6 V cm(-1) for 4 s, 117.6 V cm(-1) for 2 s and 470.4V cm(-1) for 8 s). The ME-PFSG method was found to be 4 times faster than the method using a constant field and 138 times faster than slab gel electrophoresis. Moreover, the amplified RT-PCR products of the obesity-related coenzymes in C57BL/6J mice were analyzed using only sub-micro liter samples. PMID:18539180

  20. L-Carnitine, but not coenzyme Q10, enhances the anti-osteoporotic effect of atorvastatin in ovariectomized rats

    PubMed Central

    Murad, Hussam A. S.

    2016-01-01

    Objective: Statins’ therapy in osteoporosis can aggravate muscle damage. This study was designed to assess which agent, L-carnitine or coenzyme Q10, could enhance the anti-osteoporotic effect of atorvastatin while antagonizing myopathy in ovariectomized rats. Methods: Forty-eight female Sprague Dawley rats were used; forty rats were ovariectomized while eight were sham-operated. Eight weeks post-ovariectomy, rats were divided into ovariectomized-untreated group and four ovariectomized-treated groups (n=8) which received by gavage (mg/(kg∙d), for 8 weeks) 17β-estradiol (0.1), atorvastatin (50), atorvastatin (50)+L-carnitine (100), or atorvastatin (50)+coenzyme Q10 (20). At the end of therapy, bone mineral density (BMD), bone mineral content (BMC), and serum levels of bone metabolic markers (BMMs) and creatine kinase (CK) were measured. Femurs were used for studying the breaking strength and histopathological changes. Results: Treatment with atorvastatin+L-carnitine restored BMD, BMC, and bone strength to near normal levels. Estrogen therapy restored BMD and BMC to near normal levels, but failed to increase bone strength. Although atorvastatin and atorvastatin+coenzyme Q10 improved BMD, BMC, and bone strength, they failed to restore levels to normal. All treatments decreased BMMs and improved histopathological changes maximally with atorvastatin+L-carnitine which restored levels to near normal. Atorvastatin aggravated the ovariectomy-induced increase in CK level while estrogen, atorvastatin+L-carnitine, and atorvastatin+coenzyme Q10 decreased its level mainly with atorvastatin+L-carnitine which restored the level to near normal. Conclusions: Co-administration of L-carnitine, but not coenzyme Q10, enhances the anti-osteoporotic effect of atorvastatin while antagonizing myopathy in ovariectomized rats. This could be valuable in treatment of osteoporotic patients. However, further confirmatory studies are needed. PMID:26739525

  1. Antiatherogenic, hepatoprotective, and hypolipidemic effects of coenzyme Q10 in alloxan-induced type 1 diabetic rats

    PubMed Central

    Ahmadvand, Hassan; Ghasemi-Dehnoo, Maryam

    2014-01-01

    BACKGROUND Diabetes mellitus, one of the leading metabolic syndromes, accounts for highest morbidity and mortality worldwide. In this study, we examined possible protective effect of coenzyme Q10 on lipid profile, atherogenic index, and liver enzyme markers in alloxan-induced type 1 diabetic rats. METHODS A total of 30 male rats were randomly divided into three groups; group 1 as control, group 2 diabetic untreatment, and group 3 treatments with coenzyme Q10 by 15 mg/kg i.p. daily, respectively .Diabetes was induced in the second and third groups by alloxan injection subcutaneously. After 8 weeks, the levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), very low-density lipoprotein (VLDL), high density lipoprotein (HDL), atherogenic index, atherogenic coefficient, cardiac risk ratio, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) of all groups were analyzed. Data were analyzed using non-parametric Mann-Whitney test (using SPSS) and P < 0.05 was considered as significant. RESULTS Coenzyme Q10 inhibited significantly the activities of ALT (11.17%), AST (19.35%) and ALP (36.67%) and decreased FBG (21.19%), TG (37.24%), TC (17.15%), LDL (30.44%), VLDL (37.24%), atherogenic index (44.24%), atherogenic coefficient (49.69%), and cardiac risk ratio (37.97%), HDL level was significantly (33.38%) increased when treated with coenzyme Q10. CONCLUSION The findings of this study suggest that coenzyme Q10 exert beneficial effects on the lipid profile, atherogenic index, and liver enzymes activity in alloxan-induced type 1 diabetic rats. PMID:25258634

  2. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

  3. Variations in the Localization of Acetyl-Coenzyme A Synthetase in Aerobic Yeast Cells

    PubMed Central

    Klein, Harold P.; Jahnke, Linda

    1971-01-01

    In cells of Saccharomyces cerevisiae growing aerobically for 24 hr, acetyl-coenzyme A synthetase [acetate: CoA ligase (AMP), EC 6.2.1.1] was localized principally in the microsomal fraction. On density gradients, the enzyme in such cells behaved as a low-density particle, readily separable from the soluble proteins. After 48 hr of incubation, the cells showed a bimodal distribution of enzyme, with most of the activity now sedimenting with the mitochondrial fraction and only a smaller amount with the microsomal fraction. By using density gradients, two forms of synthetase were obtained from these cells: one band denser and the other band less dense than the intact mitochondria. In all preparations containing synthetase activity, appreciable levels of phospholipids were also detected. Images PMID:4102333

  4. Factors affecting the palmitoyl-coenzyme A desaturase of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Volkmann, C. M.

    1975-01-01

    The activity and stability of the palmitoyl-coenzyme A (CoA) desaturase complex of Saccharomyces cerevisiae was influenced by several factors. Cells, grown nonaerobically and then incubated with glucose, either in air or under N2, showed a marked increase in desaturase activity. Cycloheximide, added during such incubations, prevented the increase in activity, suggesting de novo synthesis. The stability of the desaturase from cells grown nonaerobically was affected by subsequent treatment of the cells; enzyme from freshly harvested cells, or from cells that were then shaken under nitrogen, readily lost activity upon washing or during density gradient analysis, whereas aerated cells, in the presence or absence of glucose, yielded stable enzyme preparations. The loss of activity in nonaerobic preparations could be reversed by adding soluble supernatant from these homogenates and could be prevented by growing the cells in the presence of palmitoleic acid and ergosterol, but not with several other lipids tested.

  5. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  6. Hemolytic uremic syndrome and rhabdomyolysis in a patient with succinate coenzyme Q reductase (complex II) deficiency.

    PubMed

    Micheletti, M V; Lavoratti, G; Gasperini, S; Donati, M A; Pela, I

    2011-07-01

    Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. Besides diarrhea-associated HUS, due to verotoxin-producing Escherichia coli, in children HUS without prodromal diarrhea may be associated with other infectious and autoimmune diseases, genetic defects of the complement-regulator alternative-pathway, and inborn errors of vitamin B12 metabolism. Rhabdomyolysis is the dissolution of skeletal muscle due to various causes, including inborn errors of metabolism. Recurrent rhabdomyolysis and HUS have been previously described in one patient with a genetic defect of oxidative phosphorylation. We report the case of a 2-year-old boy with recurrent HUS and rhabdomyolysis in whom a succinate coenzyme Q reductase (complex II) deficiency was diagnosed. We hypothesize that defects of oxidative phosphorylation could be another etiological factor in atypical HUS. PMID:21722608

  7. beta-hydroxyisobutyryl coenzyme A deacylase deficiency: a defect in valine metabolism associated with physical malformations

    SciTech Connect

    Brown, G.K.; Hunt, S.M.; Scholem, R.; Fowler, K.; Grimes, A.; Mercer, J.F.; Truscott, R.M.; Cotton, R.G.; Rogers, J.G.; Danks, D.M.

    1982-10-01

    An infant, born to parents who were first cousins had multiple physical malformations. An associated biochemical abnormality was suggested by the urinary excretion of cysteine and cysteamine conjugates of methacrylic acid. The coenzyme A (CoA) ester of this compound is an intermediate in the pathway of valine oxidation. Subsequent investigation revealed a deficiency of beta-hydroxyisobutyryl-CoA deacylase, an enzyme unique to valine metabolism. The enzyme defect results in accumulation of methacrylyl-CoA, a highly reactive compound, which readily undergoes addition reactions with free sulfhydryl groups. Tissue damage due to reactions between methacrylyl-CoA and important sulfhydryl-containing enzymes and cofactors may account for the teratogenic effects seen in this patient.

  8. Coenzyme Q(10): a novel therapeutic approach for Fibromyalgia? case series with 5 patients.

    PubMed

    Cordero, Mario D; Alcocer-Gómez, Elísabet; de Miguel, Manuel; Cano-García, Francisco Javier; Luque, Carlos M; Fernández-Riejo, Patricia; Fernández, Ana María Moreno; Sánchez-Alcazar, José Antonio

    2011-07-01

    Coenzyme Q(10) (CoQ(10)) is an essential electron carrier in the mitochondrial respiratory chain and a strong antioxidant. Low CoQ(10) levels have been detected in patients with Fibromyalgia (FM). The purpose of the present work was to assess the effect of CoQ(10) on symptoms of five patients with FM. Patients were evaluated clinically with Visual Analogical Scale of pain (VAS), and Fibromyalgia Impact Questionnaire (FIQ). Patients with CoQ(10) deficiency showed a statistically significant reduction on symptoms after CoQ(10) treatment during 9 months (300 mg/day). Determination of deficiency and consequent supplementation in FM may result in clinical improvement. Further analysis involving more scientifically rigorous methodology will be required to confirm this observation. PMID:21496502

  9. Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase.

    PubMed Central

    Buszczak, Michael; Lu, Xiaohui; Segraves, William A; Chang, Ta Yuan; Cooley, Lynn

    2002-01-01

    During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila. PMID:11973306

  10. The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase.

    PubMed

    Wongnate, Thanyaporn; Sliwa, Dariusz; Ginovska, Bojana; Smith, Dayle; Wolf, Matthew W; Lehnert, Nicolai; Raugei, Simone; Ragsdale, Stephen W

    2016-05-20

    Methyl-coenzyme M reductase, the rate-limiting enzyme in methanogenesis and anaerobic methane oxidation, is responsible for the biological production of more than 1 billion tons of methane per year. The mechanism of methane synthesis is thought to involve either methyl-nickel(III) or methyl radical/Ni(II)-thiolate intermediates. We employed transient kinetic, spectroscopic, and computational approaches to study the reaction between the active Ni(I) enzyme and substrates. Consistent with the methyl radical-based mechanism, there was no evidence for a methyl-Ni(III) species; furthermore, magnetic circular dichroism spectroscopy identified the Ni(II)-thiolate intermediate. Temperature-dependent transient kinetics also closely matched density functional theory predictions of the methyl radical mechanism. Identifying the key intermediate in methanogenesis provides fundamental insights to develop better catalysts for producing and activating an important fuel and potent greenhouse gas. PMID:27199421

  11. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase modulator: toward age- and sex-personalized medicine.

    PubMed

    Pallottini, Valentina

    2015-01-01

    Cholesterol homeostasis maintenance is regulated by a cellular feedback system that senses cholesterol amount in cellular membranes. 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR) plays a pivotal role in cholesterol metabolism as it is the key rate-limiting enzyme of its biosynthetic pathway; its inhibition provokes a feedback response capable of reducing plasma cholesterol content. HMGR inhibition is a keystone in the treatment and prevention of cardiovascular disease and, therefore, statins (HMGR inhibitors) are widely prescribed even though they may sometimes induce side effects. These drugs are prescribed indifferently to both man and women even if there are several well-known differences in cholesterol metabolism depending on the gender and the age. Thus, gender-related differences in cholesterol metabolism should be taken into account to identify new targets for customized pharmacological treatments for hypercholesterolemia. PMID:26135220

  12. Prolonged QTc interval in association with medium-chain acyl-coenzyme A dehydrogenase deficiency.

    PubMed

    Wiles, Jason R; Leslie, Nancy; Knilans, Timothy K; Akinbi, Henry

    2014-06-01

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid oxidation. We report a term male infant who presented at 3 days of age with hypoglycemia, compensated metabolic acidosis, hypocalcemia, and prolonged QTc interval. Pregnancy was complicated by maternal premature atrial contractions and premature ventricular contractions. Prolongation of the QTc interval resolved after correction of metabolic derangements. The newborn screen was suggestive for MCAD deficiency, a diagnosis that was confirmed on genetic analysis that showed homozygosity for the disease-associated missense A985G mutation in the ACADM gene. This is the first report of acquired prolonged QTc in a neonate with MCAD deficiency, and it suggests that MCAD deficiency should be considered in the differential diagnoses of acute neonatal illnesses associated with electrocardiographic abnormality. We review the clinical presentation and diagnosis of MCAD deficiency in neonates. PMID:24799540

  13. Ockham's razor gone blunt: coenzyme Q adaptation and redox balance in tropical reef fishes

    PubMed Central

    Gagliano, Monica; Dunlap, Walter C.; de Nys, Rocky; Depczynski, Martial

    2009-01-01

    The ubiquitous coenzyme Q (CoQ) is a powerful antioxidant defence against cellular oxidative damage. In fishes, differences in the isoprenoid length of CoQ and its associated antioxidant efficacy have been proposed as an adaptation to different thermal environments. Here, we examine this broad contention by a comparison of the CoQ composition and its redox status in a range of coral reef fishes. Contrary to expectations, most species possessed CoQ8 and their hepatic redox balance was mostly found in the reduced form. These elevated concentrations of the ubiquinol antioxidant are indicative of a high level of protection required against oxidative stress. We propose that, in contrast to the current paradigm, CoQ variation in coral reef fishes is not a generalized adaptation to thermal conditions, but reflects species-specific ecological habits and physiological constraints associated with oxygen demand. PMID:19324638

  14. The Diverse Roles of Flavin Coenzymes - Nature’s Most Versatile Thespians

    PubMed Central

    Mansoorabadi, Steven O.; Thibodeaux, Christopher J.; Liu, Hung-wen

    2008-01-01

    Flavin coenzymes play a variety of roles in biological systems. This Perspective highlights the chemical versatility of flavins by reviewing research on five flavoenzymes that have been studied in our laboratory. Each of the enzymes discussed in this review (the acyl-CoA dehydrogenases (ACDs), CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), CDP-4-aceto-3,6-dideoxygalactose synthase (YerE), UDP-galactopyranose mutase (UGM), and type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2)) utilize flavin in a distinct role. In particular, the catalytic mechanisms of two of these enzymes, UGM and IDI-2, may involve novel flavin chemistry. PMID:17580897

  15. Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of coenzyme A.

    PubMed

    Rothmann, Michael; Kang, MinJin; Villa, Reymundo; Ntai, Ioanna; La Clair, James J; Kelleher, Neil L; Chapman, Eli; Burkart, Michael D

    2013-04-24

    Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues. PMID:23550886

  16. Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of Coenzyme A

    PubMed Central

    Rothmann, Michael; Kang, MinJin; Villa, Reymundo; Ntai, Ioanna; La Clair, James J.; Kelleher, Neil L.; Chapman, Eli; Burkart, Michael D.

    2013-01-01

    Pantetheine, and its corresponding disulfide pantethine, play a key role in metabolism as a building block of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and non-ribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we demonstrate that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one carbon deletion from pantetheine, can rescue a mutant E. coli strain MG1655CΔpanC lacking a functional pantothenate synthetase. Using mass spectral methods, we demonstrate that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogs. PMID:23550886

  17. TD-DFT insight into photodissociation of the Co-C bond in coenzyme B12

    PubMed Central

    Liu, Hui; Kornobis, Karina; Lodowski, Piotr; Jaworska, Maria; Kozlowski, Pawel M.

    2014-01-01

    Coenzyme B12 (AdoCbl) is one of the most biologically active forms of vitamin B12, and continues to be a topic of active research interest. The mechanism of Co-C bond cleavage in AdoCbl, and the corresponding enzymatic reactions are however, not well understood at the molecular level. In this work, time-dependent density functional theory (TD-DFT) has been applied to investigate the photodissociation of coenzyme B12. To reduce computational cost, while retaining the major spectroscopic features of AdoCbl, a truncated model based on ribosylcobalamin (RibCbl) was used to simulate Co-C photodissociation. Equilibrium geometries of RibCbl were obtained by optimization at the DFT/BP86/TZVP level of theory, and low-lying excited states were calculated by TD-DFT using the same functional and basis set. The calculated singlet states, and absorption spectra were simulated in both the gas phase, and water, using the polarizable continuum model (PCM). Both spectra were in reasonable agreement with experimental data, and potential energy curves based on vertical excitations were plotted to explore the nature of Co-C bond dissociation. It was found that a repulsive 3(σCo−C → σ*Co−C) triplet state became dissociative at large Co-C bond distance, similar to a previous observation for methylcobalamin (MeCbl). Furthermore, potential energy surfaces (PESs) obtained as a function of both Co-CRib and Co-NIm distances, identify the S1 state as a key intermediate generated during photoexcitation of RibCbl, attributed to a mixture of a metal-to-ligand charge transfer (MLCT) and a σ bonding-ligand charge transfer (SBLCT) states. PMID:24790969

  18. Structure of Coenzyme A-Disulfide Reductase from Staphylococcus aureus at 1.54 Angstrom Resolution

    SciTech Connect

    Mallett,T.; Wallen, J.; Karplus, P.; Sakai, H.; Tsukihara, T.; Claiborne, A.

    2006-01-01

    Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 {angstrom}. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S{gamma} located at the flavin si face, 3.2 {angstrom} from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 {angstrom} from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

  19. TD-DFT Insight into Photodissociation of Co-C Bond in Coenzyme B12

    NASA Astrophysics Data System (ADS)

    Kozlowski, Pawel; Liu, Hui; Kornobis, Karina; Lodowski, Piotr; Jaworska, Maria

    2013-12-01

    Coenzyme B12 (AdoCbl) is one of the most biologically active forms of vitamin B12, and continues to be a topic of active research interest. The mechanism of Co-C bond cleavage in AdoCbl, and the corresponding enzymatic reactions are however, not well understood at the molecular level. In this work, time-dependent density functional theory (TD-DFT) has been applied to investigate the photodissociation of coenzyme B12. To reduce computational cost, while retaining the major spectroscopic features of AdoCbl, a truncated model based on ribosylcobalamin (RibCbl) was used to simulate Co-C photodissociation. Equilibrium geometries of RibCbl were obtained by optimization at the DFT/BP86/TZVP level of theory, and low-lying excited states were calculated by TD-DFT using the same functional and basis set. The calculated singlet states, and absorption spectra were simulated in both the gas phase, and water, using the polarizable continuum model (PCM). Both spectra were in reasonable agreement with experimental data, and potential energy curves based on vertical excitations were plotted to explore the nature of Co-C bond dissociation. It was found that a repulsive 3(σCo-C → σ*Co-C) triplet state became dissociative at large Co-C bond distance, similar to a previous observation for methylcobalamin (MeCbl). Furthermore, potential energy surfaces (PESs) obtained as a function of both Co-CRib and Co-NIm distances, identify the S1 state as a key intermediate generated during photoexcitation of RibCbl, attributed to a mixture of a MLCT (metal-to-ligand charge transfer) and a σ bonding-ligand charge transfer (SBLCT) states.

  20. Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP+

    PubMed Central

    Liu, Xin-Xin

    2015-01-01

    ABSTRACT NADP+ is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP+ negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP+ bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP+ binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP+ concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP+ at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation. IMPORTANCE Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP+-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP+ specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP+ metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs. PMID:26598367

  1. Coenzyme Q10 protects against acute consequences of experimental myocardial infarction in rats

    PubMed Central

    Eleawa, Samy M; Alkhateeb, Mahmoud; Ghosh, Sanjoy; Al-Hashem, Fahaid; Shatoor, Abdullah S; Alhejaily, Abdulmohsen; Khalil, Mohammad A

    2015-01-01

    Aim: Myocardial infarction (MI) due to sudden occlusion of a major coronary artery leads to a complex series of events that result in left ventricle (LV) impairment eventual heart failure. Therapeutic options are limited to reverse such trends post MI. The aim of this study was to compare the acute cardioprotective effects of the antioxidants, resveratrol (RES) and coenzyme Q10 (CoQ10), either individually or in combination, on infracts size, LV hemodynamics, inflammation and oxidative stress markers in rats with experimentally induced MI. Methods: Male Wistar rats were randomly divided into six groups: control without surgery, sham without occlusion, MI without antioxidants, RES pre-treated then MI (20 mg/kg, orally), CoQ10 then MI (20 mg/kg, intramuscular.), and combined RES and CoQ10 then MI with (each group n = 10). Pretreatment commenced 7 days prior to the permanent occlusion of the left anterior descending (LAD) coronary artery. Infarct area, hemodynamics, inflammation and oxidative stress markers were assessed 24 hours post-MI. Results: Compared to RES alone, CoQ10 pre-administration either by itself or in combination with RES, significantly reduced LV infarct area (57%), and normalized LV hemodynamic parameters like LVEDP (100%), LVSP (95.4%), LV +dp/dt and -dp/dt (102 and 73.1%, respectively). CoQ10 also decreased serum levels of brain natriuretic peptide (70%), and various circulating inflammatory markers like TNF-α (83.2%) and IL-6 (83.2%). Regarding oxidative stress, TBARS scores were lowered with a concurrent increase in both superoxide dismutase and glutathione peroxidase activities with CoQ10 alone or in combination with RES. Conclusion: Coenzyme Q10 protects against the acute sequelae of myocardial infarction. It profoundly reduced infarct area, inflammation and oxidative stress while normalizing LV hemodynamics post MI. PMID:26069524

  2. Smoking habits and coenzyme Q10 status in healthy European adults

    PubMed Central

    Fischer, Alexandra; Onur, Simone; Paulussen, Michael; Menke, Thomas; Döring, Frank

    2016-01-01

    Introduction Coenzyme Q10 (CoQ10) is a lipophilic endogenously synthesised antioxidant that is present in nearly all human tissues and plays an important role in mitochondrial energy production. It has been postulated that smoking has a consumptive effect on CoQ10. Material and methods To further define the relation between smoking and the serum CoQ10 status, 276 healthy volunteers aged 19 to 62 years were grouped into non-smokers (n = 113; 77 male, 36 female) and smokers (n = 163; 102 male, 61 female). Serum lipid profile was analysed by standard clinical chemistry. Coenzyme Q10 concentration and redox status were analysed by high-pressure liquid chromatography with electrochemical detection. Results Male smokers showed higher serum CoQ10 levels than female smokers. This sex-related difference was accounted for when CoQ10 was related to low-density lipoprotein (LDL) cholesterol as the main carrier of CoQ10 in the circulation. Neither LDL-adjusted CoQ10 concentration nor redox status significantly differed when smokers and non-smokers were compared. Regarding the smoking history, the number of cigarettes consumed per day did not significantly affect the CoQ10 status. Interestingly, with increasing time of smoking habit we observed increasing levels of LDL-adjusted serum CoQ10 concentration (Spearman's p < 0.002) and of the reduced form of CoQ10 (Spearman's p < 0.0001). Conclusions As an adaptive response to oxidative stress in long-term smokers an increased demand for antioxidant capacity may be covered by increasing levels of LDL-adjusted CoQ10 serum concentrations and by a concomitantly increased availability of the reduced, active form of CoQ10, possibly by induction of enzymes that are involved in converting CoQ10ox to CoQ10red. PMID:27478450

  3. Concurrent administration of coenzyme Q10 and alpha-tocopherol improves learning in aged mice.

    PubMed

    McDonald, Shelley R; Sohal, Rajindar S; Forster, Michael J

    2005-03-15

    The main purpose of this study was to determine whether supplemental intake of coenzyme Q10 (CoQ) (ubiquinone-10) or alpha-tocopherol, either alone or together, could improve brain function of aged mice, as reflected in their cognitive or psychomotor performance. Separate groups of aged mice (24 months) were administered either CoQ (123 mg/kg/day), or alpha-tocopherol acetate (200 mg/kg/day), or both, or the vehicle (soybean oil) via gavage for a period of 14 weeks. Three weeks following the initiation of these treatments, mice were given a battery of age-sensitive behavioral tests for the assessment of learning, recent memory, and psychomotor function. In a test that required the mice to rapidly identify and remember the correct arm of a T-maze, and to respond preemptively in order to avoid an electric shock, the intake of alpha-tocopherol plus CoQ resulted in more rapid learning compared to the control group. Learning was not significantly improved in the mice receiving CoQ or alpha-tocopherol alone. None of the treatments resulted in a significant improvement of psychomotor performance in the old mice. In a separate study, treatment with higher doses of CoQ alone (250 or 500 mg/kg/day) for 14 weeks failed to produce effects comparable to those of the combination of alpha-tocopherol and CoQ. The apparent interaction of CoQ and alpha-tocopherol treatments is consistent with the previous suggestion, based on biochemical studies, that coenzyme Q and alpha-tocopherol act in concert. Overall, the findings suggest that concurrent supplementation of alpha-tocopherol with CoQ is more likely to be effective as a potential treatment for age-related learning deficits than supplementation with CoQ or alpha-tocopherol alone. PMID:15721983

  4. Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

    PubMed Central

    Carter, Michael S.

    2015-01-01

    ABSTRACT Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR). IMPORTANCE Short-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling

  5. Survival transcriptome in the coenzyme Q10 deficiency syndrome is acquired by epigenetic modifications: a modelling study for human coenzyme Q10 deficiencies

    PubMed Central

    Fernández-Ayala, Daniel J M; Guerra, Ignacio; Jiménez-Gancedo, Sandra; Cascajo, Maria V; Gavilán, Angela; DiMauro, Salvatore; Hirano, Michio; Briones, Paz; Artuch, Rafael; De Cabo, Rafael; Salviati, Leonardo; Navas, Plácido

    2013-01-01

    Objectives Coenzyme Q10 (CoQ10) deficiency syndrome is a rare condition that causes mitochondrial dysfunction and includes a variety of clinical presentations as encephalomyopathy, ataxia and renal failure. First, we sought to set up what all have in common, and then investigate why CoQ10 supplementation reverses the bioenergetics alterations in cultured cells but not all the cellular phenotypes. Design Modelling study This work models the transcriptome of human CoQ10 deficiency syndrome in primary fibroblast from patients and study the genetic response to CoQ10 treatment in these cells. Setting Four hospitals and medical centres from Spain, Italy and the USA, and two research laboratories from Spain and the USA. Participants Primary cells were collected from patients in the above centres. Measurements We characterised by microarray analysis the expression profile of fibroblasts from seven CoQ10-deficient patients (three had primary deficiency and four had a secondary form) and aged-matched controls, before and after CoQ10 supplementation. Results were validated by Q-RT-PCR. The profile of DNA (CpG) methylation was evaluated for a subset of gene with displayed altered expression. Results CoQ10-deficient fibroblasts (independently from the aetiology) showed a common transcriptomic profile that promotes cell survival by activating cell cycle and growth, cell stress responses and inhibiting cell death and immune responses. Energy production was supported mainly by glycolysis while CoQ10 supplementation restored oxidative phosphorylation. Expression of genes involved in cell death pathways was partially restored by treatment, while genes involved in differentiation, cell cycle and growth were not affected. Stably demethylated genes were unaffected by treatment whereas we observed restored gene expression in either non-methylated genes or those with an unchanged methylation pattern. Conclusions CoQ10 deficiency induces a specific transcriptomic profile that promotes

  6. l-Malyl-Coenzyme A/β-Methylmalyl-Coenzyme A Lyase Is Involved in Acetate Assimilation of the Isocitrate Lyase-Negative Bacterium Rhodobacter capsulatus

    PubMed Central

    Meister, Michael; Saum, Stephan; Alber, Birgit E.; Fuchs, Georg

    2005-01-01

    Cell extracts of Rhodobacter capsulatus grown on acetate contained an apparent malate synthase activity but lacked isocitrate lyase activity. Therefore, R. capsulatus cannot use the glyoxylate cycle for acetate assimilation, and a different pathway must exist. It is shown that the apparent malate synthase activity is due to the combination of a malyl-coenzyme A (CoA) lyase and a malyl-CoA-hydrolyzing enzyme. Malyl-CoA lyase activity was 20-fold up-regulated in acetate-grown cells versus glucose-grown cells. Malyl-CoA lyase was purified 250-fold with a recovery of 6%. The enzyme catalyzed not only the reversible condensation of glyoxylate and acetyl-CoA to l-malyl-CoA but also the reversible condensation of glyoxylate and propionyl-CoA to β-methylmalyl-CoA. Enzyme activity was stimulated by divalent ions with preference for Mn2+ and was inhibited by EDTA. The N-terminal amino acid sequence was determined, and a corresponding gene coding for a 34.2-kDa protein was identified and designated mcl1. The native molecular mass of the purified protein was 195 ± 20 kDa, indicating a homohexameric composition. A homologous mcl1 gene was found in the genomes of the isocitrate lyase-negative bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum in similar genomic environments. For Streptomyces coelicolor and Methylobacterium extorquens, mcl1 homologs are located within gene clusters implicated in acetate metabolism. We therefore propose that l-malyl-CoA/β-methylmalyl-CoA lyase encoded by mcl1 is involved in acetate assimilation by R. capsulatus and possibly other glyoxylate cycle-negative bacteria. PMID:15687206

  7. Oral Coenzyme Q10 Supplementation Does Not Prevent Cardiac Alterations During a High Altitude Trek to Everest Base Camp

    PubMed Central

    Holloway, Cameron J.; Mitchell, Kay; Martin, Daniel S.; Johnson, Andrew W.; Cochlin, Lowri E.; Codreanu, Ion; Dhillon, Sundeep; Rodway, George W.; Ashmore, Tom; Levett, Denny Z.H.; Neubauer, Stefan; Montgomery, Hugh E.; Grocott, Michael P.W.; Clarke, Kieran

    2014-01-01

    Abstract Holloway, Cameron J., Andrew J. Murray, Kay Mitchell, Daniel S. Martin, Andrew W. Johnson, Lowri E. Cochlin, Ion Codreanu, Sundeep Dhillon, George W. Rodway, Tom Ashmore, Denny Z.H. Levett, Stefan Neubauer, Hugh E. Montgomery, Michael P.W. Grocott, and Kieran Clarke, on behalf of the Caudwell Xtreme Everest 2009 Investigators. Oral Coenzyme Q supplementation does not prevent cardiac alterations during a high altitude trek to Everest Base Camp. High Alt Med Biol 15:000—000, 2014.—Exposure to high altitude is associated with sustained, but reversible, changes in cardiac mass, diastolic function, and high-energy phosphate metabolism. Whilst the underlying mechanisms remain elusive, tissue hypoxia increases generation of reactive oxygen species (ROS), which can stabilize hypoxia-inducible factor (HIF) transcription factors, bringing about transcriptional changes that suppress oxidative phosphorylation and activate autophagy. We therefore investigated whether oral supplementation with an antioxidant, Coenzyme Q10, prevented the cardiac perturbations associated with altitude exposure. Twenty-three volunteers (10 male, 13 female, 46±3 years) were recruited from the 2009 Caudwell Xtreme Everest Research Treks and studied before, and within 48 h of return from, a 17-day trek to Everest Base Camp, with subjects receiving either no intervention (controls) or 300 mg Coenzyme Q10 per day throughout altitude exposure. Cardiac magnetic resonance imaging and echocardiography were used to assess cardiac morphology and function. Following altitude exposure, body mass fell by 3 kg in all subjects (p<0.001), associated with a loss of body fat and a fall in BMI. Post-trek, left ventricular mass had decreased by 11% in controls (p<0.05) and by 16% in Coenzyme Q10-treated subjects (p<0.001), whereas mitral inflow E/A had decreased by 18% in controls (p<0.05) and by 21% in Coenzyme Q10-treated subjects (p<0.05). Coenzyme Q10 supplementation did not, therefore, prevent

  8. The Protective Effects of Alpha-Lipoic Acid and Coenzyme Q10 Combination on Ovarian Ischemia-Reperfusion Injury: An Experimental Study

    PubMed Central

    Bozkurt, Mehmet Fatih; Koken, Tulay; Dogan, Nurhan; Pektaş, Mine Kanat; Baskin Embleton, Didem

    2016-01-01

    Objective. This study aims to evaluate whether alpha-lipoic acid and/or coenzyme Q10 can protect the prepubertal ovarian tissue from ischemia-reperfusion injury in an experimental rat model of ovarian torsion. Materials and Methods. Forty-two female preadolescent Wistar-Albino rats were divided into 6 equal groups randomly. The sham group had laparotomy without torsion; the other groups had torsion/detorsion procedure. After undergoing torsion, group 2 received saline, group 3 received olive oil, group 4 received alpha-lipoic acid, group 5 received coenzyme Q10, and group 6 received both alpha-lipoic acid and coenzyme Q10 orally. The oxidant-antioxidant statuses of these groups were compared using biochemical measurement of oxidized/reduced glutathione, glutathione peroxidase and malondialdehyde, pathological evaluation of damage and apoptosis within the ovarian tissue, and immunohistochemical assessment of nitric oxide synthase. Results. The left ovaries of the alpha-lipoic acid + coenzyme Q10 group had significantly lower apoptosis scores and significantly higher nitric oxide synthase content than the left ovaries of the control groups. The alpha-lipoic acid + coenzyme Q10 group had significantly higher glutathione peroxidase levels and serum malondialdehyde concentrations than the sham group. Conclusions. The combination of alpha-lipoic acid and coenzyme Q10 has beneficial effects on oxidative stress induced by ischemia-reperfusion injury related to ovarian torsion. PMID:27597986

  9. The Protective Effects of Alpha-Lipoic Acid and Coenzyme Q10 Combination on Ovarian Ischemia-Reperfusion Injury: An Experimental Study.

    PubMed

    Tuncer, Ahmet Ali; Bozkurt, Mehmet Fatih; Koken, Tulay; Dogan, Nurhan; Pektaş, Mine Kanat; Baskin Embleton, Didem

    2016-01-01

    Objective. This study aims to evaluate whether alpha-lipoic acid and/or coenzyme Q10 can protect the prepubertal ovarian tissue from ischemia-reperfusion injury in an experimental rat model of ovarian torsion. Materials and Methods. Forty-two female preadolescent Wistar-Albino rats were divided into 6 equal groups randomly. The sham group had laparotomy without torsion; the other groups had torsion/detorsion procedure. After undergoing torsion, group 2 received saline, group 3 received olive oil, group 4 received alpha-lipoic acid, group 5 received coenzyme Q10, and group 6 received both alpha-lipoic acid and coenzyme Q10 orally. The oxidant-antioxidant statuses of these groups were compared using biochemical measurement of oxidized/reduced glutathione, glutathione peroxidase and malondialdehyde, pathological evaluation of damage and apoptosis within the ovarian tissue, and immunohistochemical assessment of nitric oxide synthase. Results. The left ovaries of the alpha-lipoic acid + coenzyme Q10 group had significantly lower apoptosis scores and significantly higher nitric oxide synthase content than the left ovaries of the control groups. The alpha-lipoic acid + coenzyme Q10 group had significantly higher glutathione peroxidase levels and serum malondialdehyde concentrations than the sham group. Conclusions. The combination of alpha-lipoic acid and coenzyme Q10 has beneficial effects on oxidative stress induced by ischemia-reperfusion injury related to ovarian torsion. PMID:27597986

  10. Coenzyme F430, quantification and isotope analysis from the Eel River Basin California

    NASA Astrophysics Data System (ADS)

    Bird, L. R.; Fulton, J. M.; Dawson, K.; Orphan, V. J.; Freeman, K. H.

    2012-12-01

    Large amounts of methane are oxidized by communities of methanotrophic archaea and sulphate-reducing bacteria, preventing this greenhouse gas from reaching the atmosphere (Orphan et al., 2001; Scheller et al., 2010). Methyl-coenzyme M reductase, an enzyme traditionally associated with methanogenesis, has recently been linked to the anaerobic oxidation of methane suggesting methane oxidation follows a pathway similar to reverse methanogenesis. Coenzyme F430, a tetrapyrrole-nickel complex within the active site of methyl-coenzyme M, is used in methanogenesis and is hypothesized to play a key role in archaeal methanotrophy (Scheller et al., 2010). We recently developed a method to extract and isolate F430 from natural sediments so it can be purified for carbon and nitrogen stable isotope analysis. Sediments are extracted using an ultrasonic homogenizer, first in water (pH 7), then twice in dilute formic acid (pH 3). The combined extract is neutralized and the F430-containing fraction is isolated using Sephadex and Amberlite column chromatography. Further purification is performed using two dimensional high performance liquid chromatography, first with a reverse phase C-18 column followed by separation on a ThermoFisher Hypercarb column. F430 is then quantified using photo diode array detection with fractions collected for isotope analysis using a nano-scale elemental analyzer isotope ratio mass spectrometer (nano-EA-IRMS; Polissar et al., 2009). Compound identity and purity are confirmed using molar C:N ratios, UV absorbance and MSn detection of the parent ion (m/z 905). Here, we report F430 concentrations and isotopic data determined from active seep sediment cores from the Eel River Basin (California), a site where the anoxic oxidation of methane occurs. A spike in the concentration of F430 is observed at the 3-6 cm depth horizon corresponding with peak abundance in ANME-2/Desulfosarcina/Desulfococcus aggregate counts. Carbon isotope values of F430 are significantly

  11. Simultaneous Analysis of Major Coenzymes of Cellular Redox Reactions and Energy Using ex Vivo (1)H NMR Spectroscopy.

    PubMed

    Nagana Gowda, G A; Abell, Lauren; Lee, Chi Fung; Tian, Rong; Raftery, Daniel

    2016-05-01

    Coenzymes of cellular redox reactions and cellular energy mediate biochemical reactions fundamental to the functioning of all living cells. Despite their immense interest, no simple method exists to gain insights into their cellular concentrations in a single step. We show that a simple (1)H NMR experiment can simultaneously measure oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD(+) and NADH), oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP(+) and NADPH), and adenosine triphosphate (ATP) and its precursors, adenosine diphosphate (ADP) and adenosine monophosphate (AMP), using mouse heart, kidney, brain, liver, and skeletal muscle tissue extracts as examples. Combining 1D/2D NMR experiments, chemical shift libraries, and authentic compound data, reliable peak identities for these coenzymes have been established. To assess this methodology, cardiac NADH and NAD(+) ratios/pool sizes were measured using mouse models with a cardiac-specific knockout of the mitochondrial Complex I Ndufs4 gene (cKO) and cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (cNAMPT) as examples. Sensitivity of NAD(+) and NADH to cKO or cNAMPT was observed, as anticipated. Time-dependent investigations showed that the levels of NADH and NADPH diminish by up to ∼50% within 24 h; concomitantly, NAD(+) and NADP(+) increase proportionately; however, degassing the sample and flushing the sample tubes with helium gas halted such changes. The analysis protocol along with the annotated characteristic fingerprints for each coenzyme is provided for easy identification and absolute quantification using a single internal reference for routine use. The ability to visualize the ubiquitous coenzymes fundamental to cellular functions, simultaneously and reliably, offers a new avenue to interrogate the mechanistic details of cellular function in health and disease. PMID:27043450

  12. On the assignment of nickel oxidation states of the Ox1,Ox2 forms of methyl-coenzyme M reductase

    SciTech Connect

    Telser, J.; Horng, Y.C.; Becker, D.F.; Hoffman, B.M.; Ragsdale, S.W.

    2000-01-12

    Methyl-coenzyme M reductase (MCR) catalyzes the chemical step of methane formation by methanogenic organisms. The reaction involves the two-electron reduction of CH{sub 3}S-CoM by N-7-mercaptoheptanoylthreoinine phosphate (CoB-SH). The authors have employed 35 GHz EPR and ENDOR spectroscopy to resolve the oxidation state of Ni in ox1, ox2 and red1 forms of MCR, isolated from methanobacterium thermoautotrophicum strain Marburg and prepared as described previously.

  13. The C-terminal extension of bacterial flavodoxin-reductases: involvement in the hydride transfer mechanism from the coenzyme.

    PubMed

    Bortolotti, Ana; Sánchez-Azqueta, Ana; Maya, Celia M; Velázquez-Campoy, Adrián; Hermoso, Juan A; Medina, Milagros; Cortez, Néstor

    2014-01-01

    To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP(+) than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding. PMID:24016470

  14. Simultaneous Analysis of Major Coenzymes of Cellular Redox Reactions and Energy Using ex Vivo 1H NMR Spectroscopy

    PubMed Central

    2016-01-01

    Coenzymes of cellular redox reactions and cellular energy mediate biochemical reactions fundamental to the functioning of all living cells. Despite their immense interest, no simple method exists to gain insights into their cellular concentrations in a single step. We show that a simple 1H NMR experiment can simultaneously measure oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH), oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH), and adenosine triphosphate (ATP) and its precursors, adenosine diphosphate (ADP) and adenosine monophosphate (AMP), using mouse heart, kidney, brain, liver, and skeletal muscle tissue extracts as examples. Combining 1D/2D NMR experiments, chemical shift libraries, and authentic compound data, reliable peak identities for these coenzymes have been established. To assess this methodology, cardiac NADH and NAD+ ratios/pool sizes were measured using mouse models with a cardiac-specific knockout of the mitochondrial Complex I Ndufs4 gene (cKO) and cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (cNAMPT) as examples. Sensitivity of NAD+ and NADH to cKO or cNAMPT was observed, as anticipated. Time-dependent investigations showed that the levels of NADH and NADPH diminish by up to ∼50% within 24 h; concomitantly, NAD+ and NADP+ increase proportionately; however, degassing the sample and flushing the sample tubes with helium gas halted such changes. The analysis protocol along with the annotated characteristic fingerprints for each coenzyme is provided for easy identification and absolute quantification using a single internal reference for routine use. The ability to visualize the ubiquitous coenzymes fundamental to cellular functions, simultaneously and reliably, offers a new avenue to interrogate the mechanistic details of cellular function in health and disease. PMID:27043450

  15. Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

    PubMed Central

    Zhang, Jin; Friedrich, Peter; Pierik, Antonio J.; Martins, Berta M.

    2014-01-01

    4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S]2+ cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S]2+ cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose. PMID:25452282

  16. para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae*

    PubMed Central

    Marbois, Beth; Xie, Letian X.; Choi, Samuel; Hirano, Kathleen; Hyman, Kyle; Clarke, Catherine F.

    2010-01-01

    Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable 13C6-isotope of pABA (p- amino[aromatic-13C6]benzoic acid ([13C6]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[13C6]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [13C6]pABA or [13C6]4HB generate both 13C6-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product 13C6-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ6. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ6 quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates. PMID:20592037

  17. Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated C1 hydrocarbons in methanogenic bacteria

    SciTech Connect

    Krone, U.E.; Laufer, K.; Thauer, R.K.; Hogenkamp, H.P. )

    1989-12-26

    Corrinoids, such as aquocobalamin, methylcobalamin, and (cyanoaquo)cobinamide, catalyze the reductive dehalogenation of CCl4 with titanium(III) citrate as the electron donor. We report here that this reaction is also effectively mediated by the nickel-containing porphinoid, coenzyme F430, found in methanogenic bacteria. Chloroform, methylene chloride, methyl chloride, and methane were detected as intermediates and products. Ethane was formed in trace amounts, and several as yet unidentified nonvolatile compounds were also generated. The rate of dehalogenation decreased in the series of CCl4, CHCl3, and CH2Cl2. With coenzyme F430 as the catalyst, the reduction of CH3Cl to CH4 proceeded more than 50 times faster than with aquocobalamin. Cell suspensions of Methanosarcina barkeri were found to catalyze the reductive dehalogenation of CCl4 with CO as the electron donor (E'0 = -0.524 V). Methylene chloride was the main end product. The kinetics of CHCl3 and CH2Cl2 formation from CCl4 were similar to those with coenzyme F430 or aquocobalamin as catalysts and titanium(III) citrate as the reductant.

  18. 4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements

    NASA Technical Reports Server (NTRS)

    Church, D. L.; Galston, A. W.

    1988-01-01

    When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

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

    PubMed Central

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

    2016-01-01

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

  20. Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis

    PubMed Central

    Mkrtchyan, Garik; Aleshin, Vasily; Parkhomenko, Yulia; Kaehne, Thilo; Luigi Di Salvo, Martino; Parroni, Alessia; Contestabile, Roberto; Vovk, Andrey; Bettendorff, Lucien; Bunik, Victoria

    2015-01-01

    Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins. PMID:26212886

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

    PubMed

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

    2016-01-01

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

  2. Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

    PubMed Central

    Priefert, H; Steinbüchel, A

    1992-01-01

    The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54. Images PMID:1356967

  3. Reduction and Methyl Transfer Kinetics of the Alpha Subunit from Acetyl-Coenzyme A Synthase

    SciTech Connect

    Xiangshi Tan; Christopher Sewell; Qingwu Yang; Paul A. Lindahl

    2003-01-15

    OAK-B135 Stopped-flow was used to evaluate the methylation and reduction kinetics of the isolated alpha subunit of acetyl-Coenzyme A synthase from Moorella thermoacetica. This catalytically active subunit contains a novel Ni-X-Fe4S4 cluster and a putative unidentified n =2 redox site called D. The D-site must be reduced for a methyl group to transfer from a corrinoid-iron-sulfur protein, a key step in the catalytic synthesis of acetyl-CoA. The Fe4S4 component of this cluster is also redox active, raising the possibility that it is the D-site or a portion thereof. Results presented demonstrate that the D-site reduces far faster than the Fe4S4 component, effectively eliminating this possibility. Rather, this component may alter catalytically important properties of the Ni center. The D-site is reduced through a pathway that probably does not involve the Fe4S4 component of this active-site cluster.

  4. Development and evaluation of coenzyme Q10 loaded solid lipid nanoparticle hydrogel for enhanced dermal delivery.

    PubMed

    Korkm, Emrah; Gokce, Evren H; Ozer, Ozgen

    2013-12-01

    Coenzyme Q10 (Q10) loaded solid lipid nanoparticles (SLN) were prepared by the high speed homogenization method and incorporated into Carbopol 974P hydrogels. Compritol 888 ATO (C888) was employed as the lipid base; Poloxamer 188 (P188) and Tween 80 (Tw80) were used as surfactant and co-surfactant. Optimum particle size with narrow distribution was obtained as 152.2 nm for blank and 142.4 nm for Q10 loaded SLNs. The overall charge of loaded SLNs was -13.7 ± 1.3 mV. Q10 entrapment efficiency was 89 % and the production yield was 94 %. Transmission electron microscopy analysis provided evidence of colloidal size, spherical shape while differential scanning calorimetry analysis confirmed recrystallization of the lipid after the preparation of SLNs. Trolox equivalent antioxidant capacity (TEAC) analysis has shown that antioxidant potential of Q10 can be protected in SLNs. Rheological characteristics demonstrated that the SLN incorporating gels were shear thinning and the mechanical strength of the gels was suitable for topical application. Diffusion studies from rat abdominal skin revealed that the delivery of Q10 was doubled in SLN incorporating gels, approximately 40 μg cm-2, in comparison with gels prepared with only Q10 (not incorporated in SLNs). As a result, it can be stated that Q10-SLN loaded gels can be successful delivery systems for carrying Q10 efficiently into the skin without losing its antioxidant properties. PMID:24451076

  5. Distribution of coenzyme F420 and properties of its hydrolytic fragments.

    PubMed

    Eirich, L D; Vogels, G D; Wolfe, R S

    1979-10-01

    The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system. PMID:40952

  6. Distribution of coenzyme F420 and properties of its hydrolytic fragments.

    PubMed Central

    Eirich, L D; Vogels, G D; Wolfe, R S

    1979-01-01

    The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system. PMID:40952

  7. Protective effects of coenzyme q(10) on decreased oxidative stress resistance induced by simvastatin.

    PubMed

    Kettawan, Aikkarach; Takahashi, Takayuki; Kongkachuichai, Ratchanee; Charoenkiatkul, Somsri; Kishi, Takeo; Okamoto, Tadashi

    2007-05-01

    The effects of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), on oxidative stress resistance and the protective effects of coenzyme Q (CoQ) were investigated. When simvastatin was administered orally to mice, the levels of oxidized and reduced CoQ(9) and CoQ(10) in serum, liver, and heart, decreased significantly when compared to those of control. The levels of thiobarbituric acid reactive substances induced by Fe(2+)-ascorbate in liver and heart mitochondria also increased significantly with simvastatin. Furthermore, cultured cardiac myocytes treated with simvastatin exhibited less resistance to oxidative stress, decreased time to the cessation of spontaneous beating in response to H(2)O(2) addition, and decreased responsiveness to electrical field stimulation. These results suggested that oral administration of simvastatin suppresses the biosynthesis of CoQ, which shares the same biosynthesis pathway as cholesterol up to farnesyl pyrophosphate, thus compromising the physiological function of reduced CoQ, which possesses antioxidant activity. However, these undesirable effects induced by simvastatin were alleviated by coadministering CoQ(10) with simvastatin to mice. Simvastatin also reduced the activity of NADPH-CoQ reductase, a biological enzyme that converts oxidized CoQ to the corresponding reduced CoQ, while CoQ(10) administration improved it. These findings may also support the efficacy of coadministering CoQ(10) with statins. PMID:18398496

  8. Severe encephalopathy associated to pyruvate dehydrogenase mutations and unbalanced coenzyme Q10 content.

    PubMed

    Asencio, Claudio; Rodríguez-Hernandez, María A; Briones, Paz; Montoya, Julio; Cortés, Ana; Emperador, Sonia; Gavilán, Angela; Ruiz-Pesini, Eduardo; Yubero, Dèlia; Montero, Raquel; Pineda, Mercedes; O'Callaghan, María M; Alcázar-Fabra, María; Salviati, Leonardo; Artuch, Rafael; Navas, Plácido

    2016-03-01

    Coenzyme Q10 (CoQ10) deficiency is associated to a variety of clinical phenotypes including neuromuscular and nephrotic disorders. We report two unrelated boys presenting encephalopathy, ataxia, and lactic acidosis, who died with necrotic lesions in different areas of brain. Levels of CoQ10 and complex II+III activity were increased in both skeletal muscle and fibroblasts, but it was a consequence of higher mitochondria mass measured as citrate synthase. In fibroblasts, oxygen consumption was also increased, whereas steady state ATP levels were decreased. Antioxidant enzymes such as NQO1 and MnSOD and mitochondrial marker VDAC were overexpressed. Mitochondria recycling markers Fis1 and mitofusin, and mtDNA regulatory Tfam were reduced. Exome sequencing showed mutations in PDHA1 in the first patient and in PDHB in the second. These genes encode subunits of pyruvate dehydrogenase complex (PDH) that could explain the compensatory increase of CoQ10 and a defect of mitochondrial homeostasis. These two cases describe, for the first time, a mitochondrial disease caused by PDH defects associated with unbalanced of both CoQ10 content and mitochondria homeostasis, which severely affects the brain. Both CoQ10 and mitochondria homeostasis appears as new markers for PDH associated mitochondrial disorders. PMID:26014431

  9. Preparation, characterization and in silico modeling of biodegradable nanoparticles containing cyclosporine A and coenzyme Q10

    NASA Astrophysics Data System (ADS)

    Ankola, D. D.; Durbin, E. W.; Buxton, G. A.; Schäfer, J.; Bakowsky, U.; Kumar, M. N. V. Ravi

    2010-02-01

    Combination therapy will soon become a reality, particularly for those patients requiring poly-therapy to treat co-existing disease states. This becomes all the more important with the increasing cost, time and complexity of the drug discovery process prompting one to look at new delivery systems to increase the efficacy, safety and patient compliance of existing drugs. Along this line, we attempted to design nano-scale systems for simultaneous encapsulation of cyclosporine A (CsA) and coenzyme Q10 (CoQ10) and model their encapsulation and release kinetics. The in vitro characterization of the co-encapsulated nanoparticles revealed that the surfactant nature, concentration, external phase volume, droplet size reduction method and drug loading concentration can all influence the overall performance of the nanoparticles. The semi-quantitative solubility study indicates the strong influence of CoQ10 on CsA entrapment which was thought to be due to an increase in the lipophilicity of the overall system. The in vitro dissolution profile indicates the influence of CoQ10 on CsA release (64%) to that of individual particles of CsA, where the release is faster and higher (86%) on 18th day. The attempts to model the encapsulation and release kinetics were successful, offering a possibility to use such models leading to high throughput screening of drugs and their nature, alone or in combination for a particular polymer, if chi-parameters are understood.

  10. Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

    SciTech Connect

    Tomita, Takeo; Fushinobu, Shinya; Kuzuyama, Tomohisa; Nishiyama, Makoto . E-mail: umanis@mail.ecc.u-tokyo.ac.jp

    2006-08-25

    To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.

  11. Preformed {beta}-amyloid fibrils are destabilized by coenzyme Q{sub 10} in vitro

    SciTech Connect

    Ono, Kenjiro; Hasegawa, Kazuhiro; Naiki, Hironobu; Yamada, Masahito . E-mail: m-yamada@med.kanazawa-u.ac.jp

    2005-04-29

    Inhibition of the formation of {beta}-amyloid fibrils (fA{beta}), as well as the destabilization of preformed fA{beta} in the CNS, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that nordihydroguaiaretic acid (NDGA) and wine-related polyphenol, myricetin (Myr), inhibit fA{beta} formation from A{beta} and destabilize preformed fA{beta} in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of coenzyme Q{sub 10} (CoQ{sub 10}) on the formation, extension, and destabilization of fA{beta} at pH 7.5 at 37 deg C in vitro. We next compared the anti-amyloidogenic activities of CoQ{sub 10} with NDGA and Myr. CoQ{sub 10} dose-dependently inhibited fA{beta} formation from amyloid {beta}-peptide (A{beta}), as well as their extension. Moreover, it destabilized preformed fA{beta}s. The anti-amyloidogenic effects of CoQ{sub 10} were slightly weaker than those of NDGA and Myr. CoQ{sub 10} could be a key molecule for the development of therapeutics for AD.

  12. Investigation of pyridine carboxylic acids in CM2 carbonaceous chondrites: Potential precursor molecules for ancient coenzymes

    NASA Astrophysics Data System (ADS)

    Smith, Karen E.; Callahan, Michael P.; Gerakines, Perry A.; Dworkin, Jason P.; House, Christopher H.

    2014-07-01

    The distribution and abundances of pyridine carboxylic acids (including nicotinic acid) in eight CM2 carbonaceous chondrites (ALH 85013, DOM 03183, DOM 08003, EET 96016, LAP 02333, LAP 02336, LEW 85311, and WIS 91600) were investigated by liquid chromatography coupled to UV detection and high resolution Orbitrap mass spectrometry. We find that pyridine monocarboxylic acids are prevalent in CM2-type chondrites and their abundance negatively correlates with the degree of pre-terrestrial aqueous alteration that the meteorite parent body experienced. We also report the first detection of pyridine dicarboxylic acids in carbonaceous chondrites. Additionally, we carried out laboratory studies of proton-irradiated pyridine in carbon dioxide-rich ices (a 1:1 mixture) to serve as a model of the interstellar ice chemistry that may have led to the synthesis of pyridine carboxylic acids. Analysis of the irradiated ice residue shows that a comparable suite of pyridine mono- and dicarboxylic acids was produced, although aqueous alteration may still play a role in the synthesis (and ultimate yield) of these compounds in carbonaceous meteorites. Nicotinic acid is a precursor to nicotinamide adenine dinucleotide, a likely ancient molecule used in cellular metabolism in all of life, and its common occurrence in CM2 chondrites may indicate that meteorites may have been a source of molecules for the emergence of more complex coenzymes on the early Earth.

  13. Coenzyme Q biosynthesis and its role in the respiratory chain structure.

    PubMed

    Alcázar-Fabra, María; Navas, Plácido; Brea-Calvo, Gloria

    2016-08-01

    Coenzyme Q (CoQ) is a unique electron carrier in the mitochondrial respiratory chain, which is synthesized on-site by a nuclear encoded multiprotein complex. CoQ receives electrons from different redox pathways, mainly NADH and FADH2 from tricarboxylic acid pathway, dihydroorotate dehydrogenase, electron transfer flavoprotein dehydrogenase and glycerol-3-phosphate dehydrogenase that support key aspects of the metabolism. Here we explore some lines of evidence supporting the idea of the interaction of CoQ with the respiratory chain complexes, contributing to their superassembly, including respirasome, and its role in reactive oxygen species production in the mitochondrial inner membrane. We also review the current knowledge about the involvement of mitochondrial genome defects and electron transfer flavoprotein dehydrogenase mutations in the induction of secondary CoQ deficiency. This mechanism would imply specific interactions coupling CoQ itself or the CoQ-biosynthetic apparatus with the respiratory chain components. These interactions would regulate mitochondrial CoQ steady-state levels and function. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26970214

  14. The Influence of Environment on the Reactivity, Dynamics and Spectroscopy of B12 Coenzymes.

    NASA Astrophysics Data System (ADS)

    Sension, Roseanne; Ahmasi Harris, D.; Carroll, Elizabeth; Stickrath, Andrew

    2006-03-01

    Adenosylcobalamin (AdoCbl) dependent enzymes catalyze a variety of chemically difficult reactions that proceed by mechanisms involving organic radicals. In these enzymes radicals are initially generated by homolysis of the cobalt-carbon bond to produce an adenosyl radical and a cob(II)alamin radical. This radical pair may also be generated by optical excitation of the AdoCbl cofactor with visible light. In the work presented here, time-resolved spectroscopic measurements spanning the time range from 10 fs to 10 ns are used to investigate the energetics and dynamics of AdoCbl and other cobalamins as a function of environment. These studies probe the influence of environment on the energy of the low-lying charge transfer states of the cobalamin and on the barriers for dissociation and for recombination of the geminate radical pair. When the AdoCbl coenzyme is bound to the enzyme glutamate mutase, the protein environment is found to stabilize the charge transfer state of AdoCbl relative to observations in water and ethylene glycol. However, the intrinsic rate constant for recombination is only slightly smaller than the rate constant measured in free solution, suggesting the protein does not greatly perturb the ground state stability of the cobalt-carbon bond.

  15. Coenzyme Q10: Is There a Clinical Role and a Case for Measurement?

    PubMed Central

    Molyneux, Sarah L; Young, Joanna M; Florkowski, Christopher M; Lever, Michael; George, Peter M

    2008-01-01

    Coenzyme Q10 (CoQ10) is an essential cofactor in the mitochondrial electron transport pathway, and is also a lipid-soluble antioxidant. It is endogenously synthesised via the mevalonate pathway, and some is obtained from the diet. CoQ10 supplements are available over the counter from health food shops and pharmacies. CoQ10 deficiency has been implicated in several clinical disorders, including but not confined to heart failure, hypertension, Parkinson’s disease and malignancy. Statin, 3-hydroxy-3- methyl-glutaryl (HMG)-CoA reductase inhibitor therapy inhibits conversion of HMG-CoA to mevalonate and lowers plasma CoQ10 concentrations. The case for measurement of plasma CoQ10 is based on the relationship between levels and outcomes, as in chronic heart failure, where it may identify individuals most likely to benefit from supplementation therapy. During CoQ10 supplementation plasma CoQ10 levels should be monitored to ensure efficacy, given that there is variable bioavailability between commercial formulations, and known inter-individual variation in CoQ10 absorption. Knowledge of biological variation and reference change values is important to determine whether a significant change in plasma CoQ10 has occurred, whether a reduction for example following statin therapy or an increase following supplementation. Emerging evidence will determine whether CoQ10 does indeed have an important clinical role and in particular, whether there is a case for measurement. PMID:18787645

  16. Functional Analyses of Two Acetyl Coenzyme A Synthetases in the Ascomycete Gibberella zeae ▿ †

    PubMed Central

    Lee, Seunghoon; Son, Hokyoung; Lee, Jungkwan; Min, Kyunghun; Choi, Gyung Ja; Kim, Jin-Cheol; Lee, Yin-Won

    2011-01-01

    Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi. PMID:21666077

  17. Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation.

    PubMed

    Wu, Fang; Gehring, Heinz

    2009-02-01

    Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC(50) up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CH(X))(4)-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO. PMID:18922879

  18. Coenzyme Q10-Binding/Transfer Protein Saposin B also Binds gamma-Tocopherol.

    PubMed

    Jin, Guangzhi; Horinouchi, Ryo; Sagawa, Tomofumi; Orimo, Nobutsune; Kubo, Hiroshi; Yoshimura, Shinichi; Fujisawa, Akio; Kashiba, Misato; Yamamoto, Yorihiro

    2008-09-01

    gamma-Tocopherol, the major form of dietary vitamin E, is absorbed in the intestine and is secreted in chylomicrons, which are then transferred to liver lysosomes. Most gamma-tocopherol is transferred to liver microsomes and is catabolized by cytochrome p450. Due to the hydrophobicity of gamma-tocopherol, a binding and transfer protein is plausible, but none have yet been isolated and characterized. We recently found that a ubiquitous cytosolic protein, saposin B, binds and transfers coenzyme Q10 (CoQ10), which is an essential factor for ATP production and an important antioxidant. Here, we report that saposin B also binds gamma-tocopherol, but not alpha-tocopherol, as efficiently as CoQ10 at pH 7.4. At acidic pH, saposin B binds gamma-tocopherol preferentially to CoQ10 and alpha-tocopherol. Furthermore, we confirmed that saposin B selectively binds gamma-tocopherol instead of CoQ10 and alpha-tocopherol at every pH between 5.4 and 8.0 when all three lipids are competing for binding. We detected gamma-tocopherol in human saposin B monoclonal antibody-induced immunoprecipitates from human urine, although the amount of gamma-tocopherol was much smaller than that of CoQ10. These results suggest that saposin B binds and transports gamma-tocopherol in human cells. PMID:18818759

  19. NLRP3 Inflammasome Is Activated in Fibromyalgia: The Effect of Coenzyme Q10

    PubMed Central

    Alcocer-Gómez, Elísabet; Culic, Ognjen; Carrión, Angel M.; de Miguel, Manuel; Díaz-Parrado, Eduardo; Pérez-Villegas, Eva M.; Bullón, Pedro; Battino, Maurizio; Sánchez-Alcazar, José Antonio

    2014-01-01

    Abstract Aims: Fibromyalgia (FM) is a prevalent chronic pain syndrome characterized by generalized hyperalgesia associated with a wide spectrum of symptoms such as fatigue and joint stiffness. Diagnosis of FM is difficult due to the lack of reliable diagnostic biomarkers, while treatment is largely inadequate. We have investigated the role of coenzyme Q10 (CoQ10) deficiency and mitochondrial dysfunction in inflammasome activation in blood cells from FM patients, and in vitro and in vivo CoQ10 deficiency models. Results: Mitochondrial dysfunction was accompanied by increased protein expression of interleukin (IL)-1β, NLRP3 (NOD-like receptor family, pyrin domain containing 3) and caspase-1 activation, and an increase of serum levels of proinflammatory cytokines (IL-1β and IL-18). CoQ10 deficiency induced by p-aminobenzoate treatment in blood mononuclear cells and mice showed NLRP3 inflammasome activation with marked algesia. A placebo-controlled trial of CoQ10 in FM patients has shown a reduced NLRP3 inflammasome activation and IL-1β and IL-18 serum levels. Innovation: These results show an important role for the NLRP3 inflammasome in the pathogenesis of FM, and the capacity of CoQ10 in the control of inflammasome. Conclusion: These findings provide new insights into the pathogenesis of FM and suggest that NLRP3 inflammasome inhibition represents a new therapeutic intervention for the disease. Antioxid. Redox Signal. 20, 1169–1180. PMID:23886272

  20. Coenzyme Q10 Levels Are Decreased in the Cerebellum of Multiple-System Atrophy Patients

    PubMed Central

    Schottlaender, Lucia V.; Bettencourt, Conceição; Kiely, Aoife P.; Chalasani, Annapurna; Neergheen, Viruna; Holton, Janice L.; Hargreaves, Iain; Houlden, Henry

    2016-01-01

    Background The objective of this study was to evaluate whether the levels of coenzyme Q10 (CoQ10) in brain tissue of multiple system atrophy (MSA) patients differ from those in elderly controls and in patients with other neurodegenerative diseases. Methods Flash frozen brain tissue of a series of 20 pathologically confirmed MSA patients [9 olivopontocerebellar atrophy (OPCA) type, 6 striatonigral degeneration (SND) type, and 5 mixed type] was used for this study. Elderly controls (n = 37) as well as idiopathic Parkinson's disease (n = 7), dementia with Lewy bodies (n = 20), corticobasal degeneration (n = 15) and cerebellar ataxia (n = 18) patients were used as comparison groups. CoQ10 was measured in cerebellar and frontal cortex tissue by high performance liquid chromatography. Results We detected a statistically significant decrease (by 3–5%) in the level of CoQ10 in the cerebellum of MSA cases (P = 0.001), specifically in OPCA (P = 0.001) and mixed cases (P = 0.005), when compared to controls as well as to other neurodegenerative diseases [dementia with Lewy bodies (P<0.001), idiopathic Parkinson's disease (P<0.001), corticobasal degeneration (P<0.001), and cerebellar ataxia (P = 0.001)]. Conclusion Our results suggest that a perturbation in the CoQ10 biosynthetic pathway is associated with the pathogenesis of MSA but the mechanism behind this finding remains to be elucidated. PMID:26894433

  1. Coenzyme Q10 and α-tocopherol reversed age-associated functional impairments in mice

    PubMed Central

    Shetty, Ritu A.; Ikonne, Uzoma S.; Forster, Michael J.; Sumien, Nathalie

    2014-01-01

    The purpose of this study was to determine if intake of the antioxidants coenzyme Q10 (CoQ10) or α-tocopherol (Toc), either alone or in combination, could ameliorate cognitive and psychomotor impairments of aged mice, as well as reduce oxidative burden in tissues. For a period of 10 weeks, male C57BL/6J mice (3 or 18 months) were fed either a control diet, or one of three diets supplemented with Toc, CoQ10 or their combination, and were tested for cognitive and psychomotor function. Old mice on the Toc or Toc/CoQ10 diets showed improved coordinated running performance. Mice on the diet containing Toc/CoQ10 demonstrated improved performance in the discriminated avoidance task. CoQ10 and Toc alone also resulted in improved performance, albeit to a lesser degree. Protein damage was decreased especially when the mice received Toc + CoQ10 combination. Overall, these results suggest that, Toc and CoQ supplementation can ameliorate age-related impairment and reduce protein oxidation. Moreover, concurrent supplementation of CoQ10 and Toc may be more effective than either antioxidant alone. PMID:25149567

  2. Strategies for regeneration of nicotinamide coenzymes emphasizing self-sufficient closed-loop recycling systems.

    PubMed

    Hummel, Werner; Gröger, Harald

    2014-12-10

    Biocatalytic reduction reactions depending on nicotinamide coenzymes require an additional reaction to regenerate the consumed cofactor. For preparative application the preferred method is the simultaneous coupling of an in situ regeneration reaction. There are different strategically advantageous routes to achieve this goal. The standard method uses a second enzyme and a second co-substrate, for example formate and formate dehydrogenase or glucose and glucose dehydrogenase. Alternatively, a second substrate is employed which is converted by the same enzyme used for the primary reaction. For example, alcohol dehydrogenase catalyzed reactions are often coupled with excess 2-propanol which is oxidized to acetone during the regeneration of NAD(P)H. A third method utilizes a reaction-internal sequence by the direct coupling of an oxidizing and a reducing enzyme reaction. Neither an additional substrate nor a further regenerating enzyme are required for the recycling reaction. This kind of "closed-loop" or "self-sufficient" redox process for cofactor regeneration has been used rarely so far. Its most intriguing advantage is that even redox reactions with unstable precursors can be realized provided that this compound is produced in situ by an opposite redox reaction. This elegant method is applicable in special cases only but increasing numbers of examples have been published during the last years. PMID:25102236

  3. Stability of ubiquinol-10 (reduced form of coenzyme Q10 ) in human blood.

    PubMed

    Matsuo, Kazuhiko; Kasai, Kazuaki; Hosoe, Kazunori; Funahashi, Iwao

    2016-04-01

    The ratio of ubiquinol-10 in total coenzyme Q10 (TQ10 ) in human plasma has been proposed as a useful biomarker of oxidative stress. Since ubiquinol-10 is easily oxidized in air, it is necessary to perform suitable processing at medical institutions prior to analysis. To establish stable storage conditions for blood to determine the ubiquinol-10/TQ10 ratios properly, the effects of temperature conditions on the stability of ubiquinol-10 were studied. Blood samples were collected from nine male Japanese volunteers. Changes in ubiquinol-10/TQ10 ratios in blood samples were evaluated under three temperature conditions (room temperature, refrigerated and ice-cooled). Plasma levels of ubiquinol-10 and ubiquinone-10 were determined by an HPLC system with electrochemical detection and the ubiquinol-10/TQ10 ratios were calculated. We found that the ubiquinol-10/TQ10 ratio was stable up to 8 or 4 h when blood samples were stored in refrigerator or ice-cold container, respectively, and its decreases during these periods were <1.0%. We conclude that, in order to evaluate ubiquinol-10/TQ10 ratios, blood samples should be stored in a refrigerator or an ice-cold container, and processed for plasma separation within 4 h. PMID:26248527

  4. Coenzyme Q10 in human blood: native levels and determinants of oxidation during processing and storage.

    PubMed

    Franke, Adrian A; Morrison, Cynthia M; Bakke, Jesse L; Custer, Laurie J; Li, Xingnan; Cooney, Robert V

    2010-06-15

    Coenzyme Q10 (Q10) is present in the circulation mainly in its reduced form (ubiquinol-10; UL10), but oxidizes quickly ex vivo to ubiquinone-10 (UN10). Therefore, native UL10:UN10 ratios, used as markers of redox status and disease risk, are difficult to measure. We established an RP-(U)HPLC method with coulometric detection to measure natively circulating UL10 and UN10 concentrations by adding a ubiquinol/ubiquinone mixture as an internal standard immediately after plasma preparation. This allowed adjustment for unavoidable artificial UL10 oxidation as well as for total losses (or gains) of analytes during sample storage, processing, and analysis because the internal standards exactly paralleled the chemical behavior of Q10. This technique applied to blood (n = 13) revealed Q10 levels of 680-3300 nM with a mean UL10:UN10 ratio of 95:5, which was inversely associated with total Q10 (r=-0.69; p=0.004). The oxidation of UL10 to UN10 was equimolar, increased by O(2), and decreased by lower temperatures or various degassing methods. Although UL10 was stable in blood or when pure in organic solvents at 22 degrees C, its oxidation was catalyzed dose dependently by alpha-tocopherol and butylated hydroxytoluene, particularly when present in combination. Key structural features for the catalytic pro-oxidant properties of phenolic antioxidants included two substituents vicinal to the phenolic hydroxyl group. PMID:20226852

  5. Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism

    PubMed Central

    Siudeja, Katarzyna; Grzeschik, Nicola A.; Rana, Anil; de Jong, Jannie; Sibon, Ody C. M.

    2012-01-01

    Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture – a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics. PMID:22912811

  6. Evaluation of Coenzyme Q as an Antioxidant Strategy for Alzheimer’s Disease

    PubMed Central

    Wadsworth, Teri L; Bishop, James A; Pappu, Anuradha S; Woltjer, Randall L; Quinn, Joseph F

    2010-01-01

    Increasing evidence suggests that Alzheimer's disease (AD) is associated with oxidative damage that is caused in part by mitochondrial dysfunction. Here we investigated the feasibility of modifying Alzheimer pathology with the mitochondrial antioxidant coenzyme Q (CoQ). Exogenous CoQ protected MC65 neuroblastoma cells from amyloid precursor protein C-terminal fragment (APP CTF)-induced neurotoxicity in a concentration dependent manner, with concentrations of 6.25 µM and higher providing near complete protection. Dietary supplementation with CoQ at a dose of 10 g/kg diet to C65/Bl6 mice for one month significantly suppressed brain protein carbonyl levels, which are markers of oxidative damage. Treatment for one month with 2 g lovastatin/kg diet, which interferes with CoQ synthesis, resulted in a significant lowering of brain CoQ10 levels. Mitochondrial energetics (brain ATP levels and mitochondrial membrane potential) were unaffected by either CoQ or lovastatin treatment. Our results suggest that oral CoQ may be a viable antioxidant strategy for neurodegenerative disease. Our data supports a trial of CoQ in an animal model of AD in order to determine whether a clinical trial is warranted. PMID:18560133

  7. Coenzyme Q10 Abrogated the 28 Days Aluminium Chloride Induced Oxidative Changes in Rat Cerebral Cortex

    PubMed Central

    Majumdar, Anuradha S.; Nirwane, Abhijit; Kamble, Rahul

    2014-01-01

    Objective: The present study was designed to elucidate the impact of oral administration of aluminium chloride for 28 days with respect to oxidative stress in the cerebral cortex of female rats. Further, to investigate the potentials of Coenzyme (Co) Q10 (4, 8, and 12 mg/kg, i.p.) in mitigating the detrimental changes. Materials and Methods: Biochemical estimations of cerebral lipid peroxidation (LPO), reduced glutathione (GSH), vitamin E and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were carried out after 28 days of aluminium chloride (AlCl3) and Co Q10 exposures along with histopathological examination of cerebral cortex of the rats. Results: Subacute exposure to AlCl3(5 mg/kg) led to significant decrease in levels of GSH, vitamin E and activities of SOD, CAT, GPx, and an increase in LPO of cerebral cortex. These aberrations were restored by Co Q10 (12 mg/kg, i.p.). This protection offered was comparable to that of L-deprenyl (1 mg/kg, i.p.) which served as a reference standard. Histopathological evaluations confirmed that the normal cerebral morphology was maintained by Co Q10. Conclusion: Thus, AlCl3 exposure hampers the activities of various antioxidant enzymes and induces oxidative stress in cerebral cortex of female Wistar rats. Supplementation with intraperitoneal Co Q10 abrogated these deleterious effects of AlCl3. PMID:25253934

  8. Can coenzyme q10 improve clinical and molecular parameters in fibromyalgia?

    PubMed

    Cordero, Mario D; Alcocer-Gómez, Elísabet; de Miguel, Manuel; Culic, Ognjen; Carrión, Angel M; Alvarez-Suarez, José Miguel; Bullón, Pedro; Battino, Maurizio; Fernández-Rodríguez, Ana; Sánchez-Alcazar, José Antonio

    2013-10-20

    Fibromyalgia (FM) is a complex disorder that affects up to 5% of the general population worldwide. Its pathophysiological mechanisms are difficult to identify and current drug therapies demonstrate limited effectiveness. Both mitochondrial dysfunction and coenzyme Q10 (CoQ10) deficiency have been implicated in FM pathophysiology. We have investigated the effect of CoQ10 supplementation. We carried out a randomized, double-blind, placebo-controlled trial to evaluate clinical and gene expression effects of forty days of CoQ10 supplementation (300 mg/day) on 20 FM patients. This study was registered with controlled-trials.com (ISRCTN 21164124). An important clinical improvement was evident after CoQ10 versus placebo treatment showing a reduction of FIQ (p<0.001), and a most prominent reduction in pain (p<0.001), fatigue, and morning tiredness (p<0.01) subscales from FIQ. Furthermore, we observed an important reduction in the pain visual scale (p<0.01) and a reduction in tender points (p<0.01), including recovery of inflammation, antioxidant enzymes, mitochondrial biogenesis, and AMPK gene expression levels, associated with phosphorylation of the AMPK activity. These results lead to the hypothesis that CoQ10 have a potential therapeutic effect in FM, and indicate new potential molecular targets for the therapy of this disease. AMPK could be implicated in the pathophysiology of FM. PMID:23458405

  9. Coenzyme Q10 depletion in medical and neuropsychiatric disorders: potential repercussions and therapeutic implications.

    PubMed

    Morris, Gerwyn; Anderson, George; Berk, Michael; Maes, Michael

    2013-12-01

    Coenzyme Q10 (CoQ10) is an antioxidant, a membrane stabilizer, and a vital cofactor in the mitochondrial electron transport chain, enabling the generation of adenosine triphosphate. It additionally regulates gene expression and apoptosis; is an essential cofactor of uncoupling proteins; and has anti-inflammatory, redox modulatory, and neuroprotective effects. This paper reviews the known physiological role of CoQ10 in cellular metabolism, cell death, differentiation and gene regulation, and examines the potential repercussions of CoQ10 depletion including its role in illnesses such as Parkinson's disease, depression, myalgic encephalomyelitis/chronic fatigue syndrome, and fibromyalgia. CoQ10 depletion may play a role in the pathophysiology of these disorders by modulating cellular processes including hydrogen peroxide formation, gene regulation, cytoprotection, bioenegetic performance, and regulation of cellular metabolism. CoQ10 treatment improves quality of life in patients with Parkinson's disease and may play a role in delaying the progression of that disorder. Administration of CoQ10 has antidepressive effects. CoQ10 treatment significantly reduces fatigue and improves ergonomic performance during exercise and thus may have potential in alleviating the exercise intolerance and exhaustion displayed by people with myalgic encepholamyletis/chronic fatigue syndrome. Administration of CoQ10 improves hyperalgesia and quality of life in patients with fibromyalgia. The evidence base for the effectiveness of treatment with CoQ10 may be explained via its ability to ameliorate oxidative stress and protect mitochondria. PMID:23761046

  10. Simultaneous analysis of ellagic acid and coenzyme Q(10) by derivative spectroscopy and HPLC.

    PubMed

    Ratnam, D Venkat; Bhardwaj, V; Kumar, M N V Ravi

    2006-09-15

    Antioxidants are gaining tremendous interest as chemopreventive as well as chemotherapeutic agents. Ellagic acid (EA) is a plant derived compound with very poor solubility in water and very low octanol/water partition coefficient and coenzyme Q(10) (CoQ(10)) is a highly lipophilic compound, which is synthesized in the body and can be derived from food supplements as well. The new insights in the combination therapy are promising a better future in many challenging diseases. Synergism is among the key advantages of combination therapy apart from decreased intensity of unwanted effects of a compound, increased patient compliance and reduction in cost of therapy. EA and CoQ(10) supplementation in combination will be beneficial in strengthening the weakened antioxidant defense system in many diseases related to oxidative stress. Here we report first derivative UV spectroscopic and HPLC methods for the simultaneous analysis of these two agents in pharmaceutical preparations. Results obtained indicate that the derivative spectroscopy is as efficient as HPLC method in quantitative analysis. Retention of ellagic acid can be increased using PEG bonded column which is poorly retained on C(18) column. PEG column can be used for rapid simultaneous analysis of EA and CoQ(10), which are having diverse physicochemical properties. PMID:18970780

  11. Application of Coenzyme Q10 for Accelerating Soft Tissue Wound Healing after Tooth Extraction in Rats

    PubMed Central

    Yoneda, Toshiki; Tomofuji, Takaaki; Kawabata, Yuya; Ekuni, Daisuke; Azuma, Tetsuji; Kataoka, Kota; Kunitomo, Muneyoshi; Morita, Manabu

    2014-01-01

    Accelerating wound healing after tooth extraction is beneficial in dental treatment. Application of antioxidants, such as reduced coenzyme Q10 (rCoQ10), may promote wound healing after tooth extraction. In this study, we examined the effects of topical application of rCoQ10 on wound healing after tooth extraction in rats. After maxillary first molars were extracted, male Fischer 344 rats (8 weeks old) (n = 27) received topical application of ointment containing 5% rCoQ10 (experimental group) or control ointment (control group) to the sockets for 3 or 8 days (n = 6–7/group). At 3 days after extraction, the experimental group showed higher collagen density and lower numbers of polymorphonuclear leukocytes in the upper part of socket, as compared to the control group (p < 0.05). Gene expression of interleukin-1β, tumor necrosis factor-α and nuclear factor-κB were also lower in the experimental group than in the control group (p < 0.05). At 8 days after tooth extraction, there were no significant differences in collagen density, number of polymorphonuclear leukocytes and bone fill between the groups. Our results suggest that topical application of rCoQ10 promotes wound healing in the soft tissue of the alveolar socket, but that rCoQ10 has a limited effect on bone remodeling in rats. PMID:25514392

  12. Coenzyme Q and Its Role in the Dietary Therapy against Aging.

    PubMed

    Varela-López, Alfonso; Giampieri, Francesca; Battino, Maurizio; Quiles, José L

    2016-01-01

    Coenzyme Q (CoQ) is a naturally occurring molecule located in the hydrophobic domain of the phospholipid bilayer of all biological membranes. Shortly after being discovered, it was recognized as an essential electron transport chain component in mitochondria where it is particularly abundant. Since then, more additional roles in cell physiology have been reported, including antioxidant, signaling, death prevention, and others. It is known that all cells are able to synthesize functionally sufficient amounts of CoQ under normal physiological conditions. However, CoQ is a molecule found in different dietary sources, which can be taken up and incorporated into biological membranes. It is known that mitochondria have a close relationship with the aging process. Additionally, delaying the aging process through diet has aroused the interest of scientists for many years. These observations have stimulated investigation of the anti-aging potential of CoQ and its possible use in dietary therapies to alleviate the effects of aging. In this context, the present review focus on the current knowledge and evidence the roles of CoQ cells, its relationship with aging, and possible implications of dietary CoQ in relation to aging, lifespan or age-related diseases. PMID:26999099

  13. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle

    PubMed Central

    Kerfeld, Cheryl A.

    2016-01-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  14. Plasma and hepatic carnitine and coenzyme A pools in a patient with fatal, valproate induced hepatotoxicity.

    PubMed Central

    Krähenbühl, S; Mang, G; Kupferschmidt, H; Meier, P J; Krause, M

    1995-01-01

    Reduced hepatic mitochondrial beta-oxidation and changes in the plasma carnitine pool are important biochemical findings in valproate induced liver toxicity. The carnitine pools in plasma and liver and the liver coenzyme A (CoA) pool in a patient with fatal, valproate induced hepatotoxicity were measured. In plasma and liver the free and total carnitine contents were decreased, whereas the ratios short chain acylcarnitine/total acid soluble carnitine were increased. The long chain acylcarnitine content was unchanged in plasma, and increased in liver. The total CoA content in liver was decreased by 84%. This was due to reduced concentrations of CoASH, acetyl-CoA, and long chain acyl-CoA whereas the concentrations of succinyl-CoA and propionyl-CoA were both increased. The good agreement between the plasma and liver carnitine pools reflects the close relation between these two pools. The observed decrease in the hepatic CoASH and total CoA content has so far not been reported in humans with valproate induced hepatotoxicity and may be functionally significant. PMID:7672665

  15. Recovery of MERRF fibroblasts and cybrids pathophysiology by coenzyme Q10.

    PubMed

    De la Mata, Mario; Garrido-Maraver, Juan; Cotán, David; Cordero, Mario D; Oropesa-Ávila, Manuel; Izquierdo, Lourdes Gómez; De Miguel, Manuel; Lorite, Juan Bautista; Infante, Eloy Rivas; Ybot, Patricia; Jackson, Sandra; Sánchez-Alcázar, José A

    2012-04-01

    Mitochondrial DNA mutations are an important cause of human disease for which there is no effective treatment. Myoclonic epilepsy with ragged-red fibers (MERRF) is a mitochondrial disease usually caused by point mutations in transfer RNA genes encoded by mitochondrial DNA. The most common mutation associated with MERRF syndrome, m.8344A > G in the gene MT-TK, which encodes transfer RNA(Lysine), affects the translation of all mitochondrial DNA encoded proteins. This impairs the assembly of the electron transport chain complexes leading to decreased mitochondrial respiratory function. Here we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from patients harboring the A8344G mutation. Coenzyme Q10 levels, as well as mitochondrial respiratory chain activity, and mitochondrial protein expression levels were significantly decreased in MERRF fibroblasts. Mitotracker staining and imaging analysis of individual mitochondria indicated the presence of small, rounded, depolarized mitochondria in MERRF fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and increased degradation of impaired mitochondria by mitophagy. Transmitochondrial cybrids harboring the A8344G mutation also showed CoQ10 deficiency, mitochondrial dysfunction, and increased mitophagy activity. All these abnormalities in patient-derived fibroblasts and cybrids were partially restored by CoQ10 supplementation, indicating that these cell culture models may be suitable for screening and validation of novel drug candidates for MERRF disease. PMID:22354625

  16. Investigation of Pyridine Carboxylic Acids in CM2 Carbonaceous Chondrites: Potential Precursor Molecules for Ancient Coenzymes

    NASA Technical Reports Server (NTRS)

    Smith, Karen E.; Callahan, Michael P.; Gerakines, Perry A.; Dworkin, Jason P.; House, Christopher H.

    2014-01-01

    The distribution and abundances of pyridine carboxylic acids (including nicotinic acid) in eight CM2 carbonaceous chondrites (ALH 85013, DOM 03183, DOM 08003, EET 96016, LAP 02333, LAP 02336, LEW 85311, and WIS 91600) were investigated by liquid chromatography coupled to UV detection and high resolution Orbitrap mass spectrometry. We find that pyridine monocarboxylic acids are prevalent in CM2-type chondrites and their abundance negatively correlates with the degree of pre-terrestrial aqueous alteration that the meteorite parent body experienced. We lso report the first detection of pyridine dicarboxylic acids in carbonaceous chondrites. Additionally, we carried out laboratory studies of proton-irradiated pyridine in carbon dioxide-rich ices (a 1:1 mixture) to serve as a model of the interstellar ice chemistry that may have led to the synthesis of pyridine carboxylic acids. Analysis of the irradiated ice residue shows that a comparable suite of pyridine mono- and dicarboxylic acids was produced, although aqueous alteration may still play a role in the synthesis (and ultimate yield) of these compounds in carbonaceous meteorites. Nicotinic acid is a precursor to nicotinamide adenine dinucleotide, a likely ancient molecule used in cellular metabolism in all of life, and its common occurrence in CM2 chondrites may indicate that meteorites may have been a source of molecules for the emergence of more complex coenzymes on the early Earth.

  17. Investigation of Pyridine Carboxylic Acids in CM2 Carbonaceous Chondrites: Potential Precursor Molecules for Ancient Coenzymes

    NASA Technical Reports Server (NTRS)

    Smith, Karen E.; Callahan, Michael P.; Gerakines, Perry A.; Dworkin, Jason P.; House, Christopher H.

    2014-01-01

    The distribution and abundances of pyridine carboxylic acids (including nicotinic acid) in eight CM2 carbonaceous chondrites (ALH 85013, DOM 03183, DOM 08003, EET 96016, LAP 02333, LAP 02336, LEW 85311, and WIS 91600) were investigated by liquid chromatography coupled to UV detection and high resolution Orbitrap mass spectrometry. We find that pyridine monocarboxylic acids are prevalent in CM2-type chondrites and their abundance negatively correlates with the degree of pre-terrestrial aqueous alteration that the meteorite parent body experienced. We also report the first detection of pyridine dicarboxylic acids in carbonaceous chondrites. Additionally, we carried out laboratory studies of proton-irradiated pyridine in carbon dioxide-rich ices (a 1:1 mixture) to serve as a model of the interstellar ice chemistry that may have led to the synthesis of pyridine carboxylic acids. Analysis of the irradiated ice residue shows that a comparable suite of pyridine mono- and dicarboxylic acids was produced, although aqueous alteration may still play a role in the synthesis (and ultimate yield) of these compounds in carbonaceous meteorites. Nicotinic acid is a precursor to nicotinamide adenine dinucleotide, a likely ancient molecule used in cellular metabolism in all of life, and its common occurrence in CM2 chondrites may indicate that meteorites may have been a source of molecules for the emergence of more complex coenzymes on the early Earth.

  18. Nano-encapsulation of coenzyme Q10 using octenyl succinic anhydride modified starch.

    PubMed

    Cheuk, Sherwin Y; Shih, Frederick F; Champagne, Elaine T; Daigle, Kim W; Patindol, James A; Mattison, Christopher P; Boue, Stephen M

    2015-05-01

    Octenyl succinic anhydride modified starch (OSA-ST) was used to encapsulate coenzyme Q10 (CoQ10). CoQ10 was dissolved in rice bran oil and incorporated into an aqueous OSA-ST solution. High pressure homogenisation of the mixture was conducted at 170 MPa for 56 cycles. The resulting emulsion had a particle size range of 200-300 nm and the absolute zeta potential varied between 8.4 and 10.6 mV. CoQ10 retention of the emulsion and freeze dried products, determined by a hexane rinse, was 98.2%. Reconstitution of the freeze dried product in Mcllvaine citrate-phosphate buffers with pH values of 3-5 and temperatures at 4 and 25 °C had very little effect on the range and distribution of the nanoparticles' size. The inflection point of the zeta potential and pH plot occurred at the first pKa of succinic acid (pH 4.2), indicating succinate as the main influence over zeta potential. PMID:25529723

  19. Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

    SciTech Connect

    Krishnakumar, A.M.; Sliwa, D.; Endrizzi, J.A.; Boyd, E.S.; Ensign, S.A.; Peters, J.W.

    2009-05-20

    Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO{sub 2} to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO{sub 2} to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

  20. Accelerated Regeneration of ATP Level after Irradiation in Human Skin Fibroblasts by Coenzyme Q10.

    PubMed

    Schniertshauer, Daniel; Müller, Sonja; Mayr, Tobias; Sonntag, Tanja; Gebhard, Daniel; Bergemann, Jörg

    2016-05-01

    Human skin is exposed to a number of harmful agents of which the ultraviolet (UV) component of solar radiation is most important. UV-induced damages include direct DNA lesions as well as oxidative damage in DNA, proteins and lipids caused by reactive oxygen species (ROS). Being the main site of ROS generation in the cell, mitochondria are particularly affected by photostress. The resulting mitochondrial dysfunction may have negative effects on many essential cellular processes. To counteract these effects, coenzyme Q10 (CoQ10 ) is used as a potent therapeutic in a number of diseases. We analyzed the mitochondrial respiration profile, the mitochondrial membrane potential and cellular ATP level in skin fibroblasts after irradiation. We observed an accelerated regeneration of cellular ATP level, a decrease in mitochondrial dysfunction as well as a preservation of the mitochondrial membrane potential after irradiation in human skin fibroblasts by treatment with CoQ10 . We conclude that the faster regeneration of the ATP level was achieved by a preservation of mitochondrial function by the addition of CoQ10 and that the protective effect of CoQ10 is primarily mediated via its antioxidative function. We suggest also that it might be further dependent on a stimulation of DNA repair enzymes by CoQ10 . PMID:26946184

  1. ANO10 mutations cause ataxia and coenzyme Q₁₀ deficiency.

    PubMed

    Balreira, Andrea; Boczonadi, Veronika; Barca, Emanuele; Pyle, Angela; Bansagi, Boglarka; Appleton, Marie; Graham, Claire; Hargreaves, Iain P; Rasic, Vedrana Milic; Lochmüller, Hanns; Griffin, Helen; Taylor, Robert W; Naini, Ali; Chinnery, Patrick F; Hirano, Michio; Quinzii, Catarina M; Horvath, Rita

    2014-11-01

    Inherited ataxias are heterogeneous disorders affecting both children and adults, with over 40 different causative genes, making molecular genetic diagnosis challenging. Although recent advances in next-generation sequencing have significantly improved mutation detection, few treatments exist for patients with inherited ataxia. In two patients with adult-onset cerebellar ataxia and coenzyme Q10 (CoQ10) deficiency in muscle, whole exome sequencing revealed mutations in ANO10, which encodes anoctamin 10, a member of a family of putative calcium-activated chloride channels, and the causative gene for autosomal recessive spinocerebellar ataxia-10 (SCAR10). Both patients presented with slowly progressive ataxia and dysarthria leading to severe disability in the sixth decade. Epilepsy and learning difficulties were also present in one patient, while retinal degeneration and cataract were present in the other. The detection of mutations in ANO10 in our patients indicate that ANO10 defects cause secondary low CoQ10 and SCAR10 patients may benefit from CoQ10 supplementation. PMID:25182700

  2. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

    PubMed

    Erbilgin, Onur; Sutter, Markus; Kerfeld, Cheryl A

    2016-03-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  3. The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini

    PubMed Central

    Demirev, Atanas V.; Khanal, Anamika; Sedai, Bhishma R.; Lim, Si Kyu; Na, Min Kyun

    2010-01-01

    Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of ε subunit. The accA3 encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2587-2) contains supplementary material, which is available to authorized users. PMID:20437235

  4. In Vivo Analysis of Folate Coenzymes and Their Compartmentation in Saccharomyces Cerevisiae

    PubMed Central

    McNeil, J. B.; Bognar, A. L.; Pearlman, R. E.

    1996-01-01

    In eukaryotes, enzymes responsible for the interconversion of one-carbon units exist in parallel in both mitochondria and the cytoplasm. Strains of Saccharomyces cerevisiae were constructed that possess combinations of gene disruptions at the SHM1 [mitochondrial serine hydroxymethyltransferase (SHMTm)], SHM2 [cytoplasmic SHMT (SHMTc)], MIS1 [mitochondrial C(1)-tetrahydrofolate synthase (C(1)-THFSm)], ADE3 [cytoplasmic C(1)-THF synthase (C(1)-THFSc)], GCV1 [glycine cleavage system (GCV) protein T], and the GLY1 (involved in glycine synthesis) loci. Analysis of the in vivo growth characteristics and phenotypes was used to determine the contribution to cytoplasmic nucleic acid and amino acid anabolism by the mitochondrial enzymes involved in the interconversion of folate coenzymes. The data indicate that mitochondria transport formate to the cytoplasmic compartment and mitochondrial synthesis of formate appears to rely primarily on SHMTm rather than the glycine cleavage system. The glycine cleavage system and SHMTm cooperate to specifically synthesize serine. With the inactivation of SHM1, however, the glycine cleavage system can make an observable contribution to the level of mitochondrial formate. Inactivation of SHM1, SHM2 and ADE3 is required to render yeast auxotrophic for TMP and methionine, suggesting that TMP synthesized in mitochondria may be available to the cytoplasmic compartment. PMID:8852837

  5. Dependence of brown adipose tissue function on CD36-mediated coenzyme Q uptake.

    PubMed

    Anderson, Courtney M; Kazantzis, Melissa; Wang, Jinshan; Venkatraman, Subramaniam; Goncalves, Renata L S; Quinlan, Casey L; Ng, Ryan; Jastroch, Martin; Benjamin, Daniel I; Nie, Biao; Herber, Candice; Van, An-Angela Ngoc; Park, Michael J; Yun, Dawee; Chan, Karen; Yu, Angela; Vuong, Peter; Febbraio, Maria; Nomura, Daniel K; Napoli, Joseph L; Brand, Martin D; Stahl, Andreas

    2015-02-01

    Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells synthesize CoQ mostly endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function are not well understood. Here, we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective nonshivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis. PMID:25620701

  6. [3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency as a cause of severe neurological damage].

    PubMed

    Dodelson de Kremer, R; Kelley, R I; Depetris de Boldini, C; Paschini de Capra, A; Corbella, L; Givogri, I; Giner de Ayala, A; Albarenque, M

    1992-01-01

    This paper describes the first Argentine case of 3-hydroxy-3-methylglutaric aciduria, a genetic defect of ketogenesis and leucine catabolism step. At the age of 4 months, the patient presented a life-threatening episode of hypoglucemia, metabolic acidosis and hyperammonemia resembling Reye syndrome. The lack of urinary ketone bodies, normal levels of plasma aminoacids and normal urinary excretion of p-hydroxyphenolic acids, led us to look for a ketogenic defect. An abnormal profile of urinary organic acids detected by thin layer chromatography and later characterized and quantified by gas chromatography-mass spectrometry (Figs. 1, 2; Table 1), showed a marked increase in the acidic metabolites typical of the 3-hydroxy-3-methylglutaric aciduria: 3-hydroxy-3-methylglutaric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxyisovaleric acids. The activity of 3-hydroxy-3-methylglutaryl coenzyme A lyase was absent in white cell pellets and between 2-5% of the control values in skin fibroblasts (Table 2). Treatment of the disorder, mainly restricted leucine or low-protein diet and addition of L-carnitine had no significant effect on the severe neurological injuries present since the first illness. MRI of the brain, at the age of 1 year and 8 months, showed images in T1 suggestive of marked cerebral atrophy and in T2 hyperintensive images predominating in the right frontal and posterior parietal areas and of the punctiform lesions in the basal ganglia, particularly in the heads of both caudate nuclei.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1302289

  7. The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

    PubMed Central

    Profant, Deborah A.; Roberts, Christopher J.; Koning, Ann J.; Wright, Robin L.

    1999-01-01

    In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain. PMID:10512876

  8. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

    PubMed Central

    Palosaari, N R; Rogers, P

    1988-01-01

    The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases. Images PMID:3384801

  9. Polyunsaturated fatty acyl-coenzyme As are inhibitors of cholesterol biosynthesis in zebrafish and mice

    PubMed Central

    Karanth, Santhosh; Tran, Vy My; Kuberan, Balagurunathan; Schlegel, Amnon

    2013-01-01

    SUMMARY Lipid disorders pose therapeutic challenges. Previously we discovered that mutation of the hepatocyte β-hydroxybutyrate transporter Slc16a6a in zebrafish causes hepatic steatosis during fasting, marked by increased hepatic triacylglycerol, but not cholesterol. This selective diversion of trapped ketogenic carbon atoms is surprising because acetate and acetoacetate can exit mitochondria and can be incorporated into both fatty acids and cholesterol in normal hepatocytes. To elucidate the mechanism of this selective diversion of carbon atoms to fatty acids, we fed wild-type and slc16a6a mutant animals high-protein ketogenic diets. We find that slc16a6a mutants have decreased activity of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), despite increased Hmgcr protein abundance and relative incorporation of mevalonate into cholesterol. These observations suggest the presence of an endogenous Hmgcr inhibitor. We took a candidate approach to identify such inhibitors. First, we found that mutant livers accumulate multiple polyunsaturated fatty acids (PUFAs) and PUFA-CoAs, and we showed that human HMGCR is inhibited by PUFA-CoAs in vitro. Second, we injected mice with an ethyl ester of the PUFA eicosapentaenoic acid and observed an acute decrease in hepatic Hmgcr activity, without alteration in Hmgcr protein abundance. These results elucidate a mechanism for PUFA-mediated cholesterol lowering through direct inhibition of Hmgcr. PMID:24057001

  10. The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis*

    PubMed Central

    Spry, Christina; Saliba, Kevin J.

    2009-01-01

    Pantothenate, a precursor of the fundamental enzyme cofactor coenzyme A (CoA), is essential for growth of the intraerythrocytic stage of human and avian malaria parasites. Avian malaria parasites have been reported to be incapable of de novo CoA synthesis and instead salvage CoA from the host erythrocyte; hence, pantothenate is required for CoA biosynthesis within the host cell and not the parasite itself. Whether the same is true of the intraerythrocytic stage of the human malaria parasite, Plasmodium falciparum, remained to be established. In this study we investigated the metabolic fate of [14C]pantothenate within uninfected and P. falciparum-infected human erythrocytes. We provide evidence consistent with normal human erythrocytes, unlike rat erythrocytes (which have been reported to possess an incomplete CoA biosynthesis pathway), being capable of CoA biosynthesis from pantothenate. We also show that CoA biosynthesis is substantially higher in P. falciparum-infected erythrocytes and that P. falciparum, unlike its avian counterpart, generates most of the CoA synthesized in the infected erythrocyte, presumably necessitated by insufficient CoA biosynthesis in the host erythrocyte. Our data raise the possibility that malaria parasites rationalize their biosynthetic activity depending on the capacity of their host cell to synthesize the metabolites they require. PMID:19584050