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Complete enzymatic oxidation of methanol to carbon dioxide: towards more eco-efficient regeneration systems for reduced nicotinamide cofactors
Kara, S. ; Schrittwieser, J.H. ; Gargiulo, S. ; Ni, Y. ; Yanase, H. ; Opperman, D.J. ; Berkel, W.J.H. van; Hollmann, F. - \ 2015
Advanced Synthesis and Catalysis 357 (2015)8. - ISSN 1615-4150 - p. 1687 - 1691.
pseudomonas-putida f61 - formaldehyde dismutase - biocatalysis - reductions - aldehydes - reductase
A novel system for in situ regeneration of reduced nicotinamide cofactors (NADH) is proposed: through a cascade of alcohol dehydrogenase (ADH), formaldehyde dismutase (FDM) and formate dehydrogenase (FDH) complete oxidation of methanol to carbon dioxide (CO2) is coupled to the regeneration of NADH. As a consequence, from one equivalent of methanol three equivalents of NADH can be obtained. The feasibility of this cascade is demonstrated at the examples of an NADH-dependent reduction of conjugated C[DOUBLE BOND]C-double bonds (catalysed by an enoate reductase) and the NADH-dependent hydroxylation of phenols (catalysed by a monooxygenase). The major limitation of the current regeneration system is the comparably poor catalytic efficiency of the methanol oxidation step (low kcat and high KM value of the ADH used) necessitating higher than theoretical methanol concentrations.
(+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways
Cankar, K. ; Jongedijk, E.J. ; Klompmaker, M. ; Majdic, T. ; Mumm, R. ; Bouwmeester, H.J. ; Bosch, H.J. ; Beekwilder, M.J. - \ 2015
Biotechnology Journal 10 (2015)1. - ISSN 1860-6768 - p. 180 - 189.
plant transformation - biosynthetic-pathway - terpenoid metabolism - squalene synthase - tobacco - expression - artemisinin - arabidopsis - reductase - precursors
Plant sesquiterpenes, such as (+)-valencene, artemisinin, and farnesene are valuable chemicals for use as aromatics, pharmaceuticals, and biofuels. Plant-based production systems for terpenoids critically depend on the availability of farnesyl diphosphate (FPP). Currently, these systems show insufficient yields, due to the competition for FPP of newly introduced pathways with endogenous ones. In this study, for the first time an RNAi strategy aiming at silencing of endogenous pathways for increased (+)-valencene production was employed. Firstly, a transient production system for (+)-valencene in Nicotiana benthamiana was set up using agroinfiltration. Secondly, silencing of the endogenous 5-epi-aristolochene synthase (EAS) and squalene synthase (SQS) that compete for the FPP pool was deployed. This resulted in a N. benthamiana plant that produces (+)-valencene as a prevalent volatile with a 2.8-fold increased yield. Finally, the size of the FPP pool was increased by overexpression of enzymes that are rate-limiting in FPP biosynthesis. Combined with silencing of EAS and SQS, no further increase of (+)-valencene production was observed, but emission of farnesol. Formation of farnesol, which is a breakdown product of FPP, indicates that overproducing sesquiterpenes is no longer limited by FPP availability in the cytosol. This study shows that metabolic engineering of plants can effectively be used for increased production of desired products in plants. Keywords: 5-Epi-aristolochene synthase · Metabolic engineering · RNAi · Squalene synthase
DNA methylation and cognitive functioning in healthy older adults
Schiepers, O.J.G. ; Boxtel, M.P.J. van; Groot, R.H.M. ; Jolles, J. ; Kok, F.J. ; Verhoef, P. ; Durga, J. - \ 2012
British Journal of Nutrition 107 (2012)5. - ISSN 0007-1145 - p. 744 - 748.
physical-activity - common mutation - homocysteine - folate - disorders - reductase - disease
Long-term supplementation with folic acid may improve cognitive performance in older individuals. The relationship between folate status and cognitive performance might be mediated by changes in methylation capacity, as methylation reactions are important for normal functioning of the brain. Although aberrant DNA methylation has been implicated in neurodevelopmental disorders, the relationship between DNA methylation status and non-pathological cognitive functioning in human subjects has not yet been investigated. The present study investigated the associations between global DNA methylation and key domains of cognitive functioning in healthy older adults. Global DNA methylation, defined as the percentage of methylated cytosine to total cytosine, was measured in leucocytes by liquid chromatography-MS/MS, in 215 men and women, aged 50-70 years, who participated in the Folic Acid and Carotid Intima-Media Thickness (FACIT) study (clinical trial registration number NCT00110604). Cognitive performance was assessed by means of the Visual Verbal Word Learning Task, the Stroop Colour-Word Interference Test, the Concept Shifting Test, the Letter-Digit Substitution Test and the Verbal Fluency Test. Using hierarchical linear regression analyses adjusted for age, sex, level of education, alcohol consumption, smoking status, physical activity, erythrocyte folate concentration and 5,10-methylenetetrahydrofolate reductase 677 C ! T genotype, we found that global DNA methylation was not related to cognitive performance on any of the domains measured. The present study results do not support the hypothesis that global DNA methylation, as measured in leucocytes, might be associated with cognitive functioning in healthy older individuals.
Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.
Ryden, A.M. ; Ruyter-Spira, C.P. ; Litjens, R. ; Takahashi, S. ; Quax, W.J. ; Osada, H. ; Bouwmeester, H.J. ; Kayser, O. - \ 2010
Plant and Cell Physiology 51 (2010)7. - ISSN 0032-0781 - p. 1219 - 1228.
chain dehydrogenases/reductases sdrs - amorpha-4,11-diene synthase - functional assignments - biosynthetic-pathway - essential oil - key enzyme - expression - reductase - peppermint - acid
From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids, and in particular functions as a menthone:neomenthol oxidoreductase. The kinetic parameter kcat/Km was determined to be 939 fold more efficient for the reduction of (-)-menthone to (+)-neomenthol, than results previously reported for the menthone:neomenthol reductase from Mentha x piperita. Based on its kinetic properties, the possible use of Red1 in biological crop protection is discussed.
Nicotiana benthamiana as a Production Platform for Artemisinin Precursors
Herpen, T.W.J.M. van; Cankar, K. ; Nogueira, M. ; Bosch, H.J. ; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2010
PLoS One 5 (2010)12. - ISSN 1932-6203 - 11 p.
antimalarial-drug artemisinin - expression system - molecular-cloning - plants - biosynthesis - annua - reductase - tobacco - yield - acid
Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-ß-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.
Purification and characterization of a chlorite dismutase from Pseudomonas chloritidismutans
Mehboob, F. ; Wolterink, A.F.W.M. ; Vermeulen, A.J. ; Jiang, B. ; Hagedoorn, P.L. ; Stams, A.J.M. ; Kengen, S.W.M. - \ 2009
FEMS Microbiology Letters 293 (2009)1. - ISSN 0378-1097 - p. 115 - 121.
desulfovibrio-vulgaris hildenborough - (per)chlorate-reducing bacteria - strain gr-1 - reductase - catalase
The chlorite dismutase (Cld) of Pseudomonas chloritidismutans was purified from the periplasmic fraction in one step by hydroxyapatite chromatography. The enzyme has a molecular mass of 110 kDa and consists of four 31-kDa subunits. Enzyme catalysis followed Michaelis-Menten kinetics, with Vmax and K(m) values of 443 U mg(-1) and 84 microM, respectively. A pyridine-NaOH-dithionite-reduced Cld revealed a Soret peak at 418 nm, indicative for protoheme IX. The spectral data indicate the presence of 1.5 mol protoheme IX mol(-1) tetrameric enzyme while metal analysis revealed 2.2 mol iron mol(-1) tetrameric enzyme. High concentrations of chlorite resulted in the disappearance of the Soret peak, which coincided with loss in activity. Electron paramagnetic resonance analyses showed an axial high-spin ferric iron signal. Cld was inhibited by cyanide, azide, but not by hydroxylamine or 3-amino-1,2,3-triazole. Remarkably, the activity was drastically enhanced by kosmotropic salts, and chaotropic salts decreased the activity, in accordance with the Hofmeister series. Chlorite conversion in the presence of 18O-labeled water did not result in the formation of oxygen with a mass of 34 (16O-18O) or a mass of 36 ((18)O-(18)O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite
Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis
Wolterink-van Loo, S. ; Levisson, M. ; Cabrières, M.C. ; Franssen, M.C.R. ; Oost, J. van der - \ 2008
Extremophiles 12 (2008)3. - ISSN 1431-0651 - p. 461 - 469.
escherichia-coli - crystal-structure - l-lysine - beta-semialdehyde - semi-aldehyde - biosynthesis - resolution - mechanism - reductase - acid
Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine¿synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T 0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-ß -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.
Differential induction of electrophile-responsive element-regulated genes by n-3 and n-6 polyunsaturated fatty acids
Beelen, V.A. van; Aarts, M.G.M. ; Reus, A. ; Mooibroek, H. ; Sijtsma, L. ; Bosch, H.J. ; Rietjens, I.M.C.M. ; Alink, G.M. - \ 2006
FEBS Letters 580 (2006)19. - ISSN 0014-5793 - p. 4587 - 4590.
antioxidant response - fish-oil - dietary - carcinogenesis - mechanisms - protection - oxidation - reductase - pathway - cancer
In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.
In vivo relevance of two critical levels for NAD(P)H:quinone oxidoreductase (NQO1)-mediated cellular protection against electrophile toxicity found in vitro
Haan, L.H.J. de; Pot, G.K. ; Aarts, J.M.M.J.G. ; Rietjens, I.M.C.M. ; Alink, G.M. - \ 2006
Toxicology in Vitro 20 (2006)5. - ISSN 0887-2333 - p. 594 - 600.
dt-diaphorase - menadione toxicity - quinone toxicity - human colon - cells - nqo1 - reductase - enzymes - lines - sensitivity
NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose¿response curve revealing a `lower protection threshold¿ of 0.5 ¿mol DCPIP/min/mg protein and an `upper protection threshold¿ at 1 ¿mol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I3C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed `lower protection threshold¿ in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this `lower protection threshold¿ will require 5¿20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds
Human NAD(P)H:Quinone oxidoreductase inhibition by flavonoids in living cells
Lee, Y.Y. ; Westphal, A.H. ; Haan, L.H.J. de; Aarts, J.M.M.J.G. ; Rietjens, I.M.C.M. - \ 2005
Free Radical Biology and Medicine 39 (2005)2. - ISSN 0891-5849 - p. 257 - 265.
hamster ovary cells - dt-diaphorase - quinone oxidoreductase - acceptor oxidoreductase - antitumor quinones - human plasma - cancer risk - quercetin - reductase - rat
Procedures for assessing enzyme inhibition in living cells are an important tool in the study of the relevance of enzyme-catalyzed reactions and interactions in the human body. This paper presents the effects of flavonoids on NAD(P)H:quinone oxidoreductase 1 (NQO1) activity, by a newly developed method to measure NQO1 inhibition in intact cells. The principle of this method is based on the resorufin reductase activity of NQO1. The change in fluorescence in time was used to determine NQO1 activity in intact Chinese hamster ovary (CHO) cells genetically engineered to overexpress human NQO1. Applying this method to determine the inhibitory effects of reported in vitro NQO1 inhibitors (dicoumarol, 7,8-dihydroxyflavone, chrysin) showed that for all inhibitors tested, the IC50 in intact cells was at least 3 orders of magnitude higher than the IC50 in cell lysates. This result demonstrates that in vitro studies with purified NQO1 or with extracts from disrupted tissues are of limited value for obtaining insight into the situation in living cells. Possible factors underlying this discrepancy are being discussed. For the first time, we determined NQO1 inhibition by flavonoids in cells without disruption of the cells or addition of cofactors, enabling the assessment of enzymatic activity and the interaction of modulators of enzymatic activity in an intracellular situation.
Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fisheri NAD(P)H:FMN-oxidoreductase
Vetrova, E.V. ; Kudryasheva, N.S. ; Visser, A.J.W.G. ; Hoek, A. van - \ 2005
Luminescence 20 (2005)3. - ISSN 1522-7235 - p. 205 - 209.
flavodoxin - anisotropy - reductase - dynamics - system
The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds
Studies on the DT-diaphorase-catalysed reaction employing quinones as substrates: evidence for a covalent modification of DT-diaphorase by tetrachloro-p-benzoquinone
Osman, A.M. ; Boeren, J.A. - \ 2004
Chemico-Biological Interactions 147 (2004)1. - ISSN 0009-2797 - p. 99 - 108.
one-electron - rat-liver - acceptor oxidoreductase - mechanism - reductase - nitrobenzimidazoles - cytotoxicity - conversion - protein - nad(p)h
In this study, the kinetic parameters, Vmax and Km, of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no correlation between the experimentally determined rates of p-benzoquinone reduction by DT-diaphorase and the calculated chemical reactivity of the examined substrates as expressed by the energy of the lowest unoccupied molecular orbital, E(LUMO). However, a reasonable correlation was found between the natural logarithm of Vmax/Km and the partition coefficient of the p-benzoquinones (r=0.81). Furthermore, tetrachloro-p-benzoquinone, one of the tested quinones is shown to be an inhibitor of rat DT-diaphorase. The presence of bovine serum albumin (BSA) in the incubation mixture protects DT-diaphorase against the inactivation by tetrachloro-p-benzoquinone, probably by interacting with the quinone. Maldi-Tof analysis of the incubation mixture of the purified DT-diaphorase and tetrachloro-p-benquinone showed that every subunit of the enzyme shifted about +414 amu, whereas the dimer shifted about +849 amu relative to control values. This indicates a covalent modification of the rat liver DT-diaphorase by tetrachloro-p-benzoquinone.
Glutathione protects Lactococcus lactis against oxidative stress
Li, Y. ; Hugenholtz, J. ; Abee, T. ; Molenaar, D. - \ 2003
Applied and Environmental Microbiology 69 (2003)10. - ISSN 0099-2240 - p. 5739 - 5745.
escherichia-coli - saccharomyces-cerevisiae - oxidized glutathione - growth-conditions - acid bacteria - reductase - thioredoxin - purification - peroxidase - metabolism
Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to similar to60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.
Metabolic control analysis of xylose catabolism in Aspergillus
Prathumpai, W. ; Gabelgaard, J.B. ; Wanchanthuek, P. ; Vondervoort, P.J.I. van de; Groot, M.J.L. de; McIntyre, M. ; Nielsen, J. - \ 2003
Biotechnology Progress 19 (2003). - ISSN 8756-7938 - p. 1136 - 1141.
yeast pichia-stipitis - nad+-xylitol-dehydrogenase - fuel ethanol-production - d-xylulokinase - l-arabinose - purification - niger - fermentation - reductase - polyol
A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out, and flux control was shown to be dependent on the metabolite levels. Due to thermodynamic constraints, flux control may reside at the first step in the pathway, i.e., at the xylose reductase, even when the intracellular xylitol concentration is high. On the basis of the kinetic analysis, the general dogma specifying that flux control often resides at the step following an intermediate present at high concentrations was, therefore, shown not to hold. The intracellular xylitol concentration was measured in batch cultivations of two different strains of Aspergillus niger and two different strains of Aspergillus nidulans grown on media containing xylose, and a concentration up to 30 mM was found. Applying MCA showed that the first polyol dehydrogenase (XDH) in the catabolic pathway of xylose exerted the main flux control in the two strains of A. nidulans and A. niger NW324, but the flux control was exerted mainly at the first enzyme of the pathway (XR) of A. niger NW 296.