Facile N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by the flavin-containing monooxygenase. A convenient synthesis of tritiated [methyl-3H]-4-phenyl-2,3-dihydropyridinium species

International Nuclear Information System (INIS)

Cashman, J.R.

1988-01-01

A rapid, efficient procedure useful for the radiosynthesis of [Me- 3 H]-MPDP+ ([methyl- 3 H]-4-phenyl-2,3-dihydropyridinium species) is described. Hog liver microsomes or the highly purified flavin-containing monooxygenase from hog liver quantitatively biotransforms [Me- 3 H]-MPTP to its corresponding radiolabeled N-oxide. For the small-scale synthesis required for radiolabeling procedures, this enzymatic process is superior to H 2 O 2 -mediated N-oxygenation of MPTP. In the presence of 0.5 mM NADPH, 4.5 mM n-octylamine, and 2 microCi [Me- 3 H]-MPTP, the only product detected in extracts from incubations performed with hog liver microsomes or purified hog liver flavin-containing monooxygenase is [Me- 3 H]-MPTP N-oxide. [Me- 3 H]-MPTP N-oxide is almost completely converted to [Me- 3 H]-MPDP+ by the action of trifluoroacetic anhydride. This procedure has the advantage of using a commercially available tritiated starting material, efficient transformations, and easily accomplished purification to afford a rapid synthesis of [Me- 3 H]-MPDP+

Flavin-containing monooxygenase 3 (FMO3) role in busulphan metabolic pathway

Science.gov (United States)

Terelius, Ylva; Abedi-Valugerdi, Manuchehr; Naughton, Seán; Saghafian, Maryam; Moshfegh, Ali; Mattsson, Jonas; Potácová, Zuzana; Hassan, Moustapha

2017-01-01

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and

Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

Science.gov (United States)

Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

2014-04-01

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

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Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2003-04-01

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

International Nuclear Information System (INIS)

Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

2008-01-01

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

Science.gov (United States)

2015-01-01

Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O2, NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H2O2. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H2O2 release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO(H) intermediate from reduced SidA and O2 is likewise independent of pH. However, the rate constants for C4a-OO(H) reactions, either to eliminate H2O2 or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H2O2 conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H2O2 release via an acid–base mechanism and where side-chain position determines whether H2O2 or the hydroxylation product is observed. PMID:24490904

An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

Science.gov (United States)

Jones, Barry C; Srivastava, Abhishek; Colclough, Nicola; Wilson, Joanne; Reddy, Venkatesh Pilla; Amberntsson, Sara; Li, Danxi

2017-10-01

Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CL int ) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CL int u ) for each compound. This was compared with observed unbound intrinsic clearance (observed CL int u ) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CL int u using hepatocytes ( R 2 = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained ( R 2 = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Inhibition of the Flavin-Dependent Monooxygenase Siderophore A (SidA) Blocks Siderophore Biosynthesis and Aspergillus fumigatus Growth.

Science.gov (United States)

Martín Del Campo, Julia S; Vogelaar, Nancy; Tolani, Karishma; Kizjakina, Karina; Harich, Kim; Sobrado, Pablo

2016-11-18

Aspergillus fumigatus is an opportunistic fungal pathogen and the most common causative agent of fatal invasive mycoses. The flavin-dependent monooxygenase siderophore A (SidA) catalyzes the oxygen and NADPH dependent hydroxylation of l-ornithine (l-Orn) to N 5 -l-hydroxyornithine in the biosynthetic pathway of hydroxamate-containing siderophores in A. fumigatus. Deletion of the gene that codes for SidA has shown that it is essential in establishing infection in mice models. Here, a fluorescence polarization high-throughput assay was used to screen a 2320 compound library for inhibitors of SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive inhibitor of SidA with a MIC value of 2 μM. Docking experiments suggest that celastrol binds across the NADPH and l-Orn pocket. Celastrol prevents A. fumigatus growth in blood agar. The addition of purified ferric-siderophore abolished the inhibitory effect of celastrol. Thus, celastrol inhibits A. fumigatus growth by blocking siderophore biosynthesis through SidA inhibiton.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

Science.gov (United States)

Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila : A Structurally Distinct Biocatalyst for Bulky Substrates

NARCIS (Netherlands)

Fürst, Maximilian J L J; Savino, Simone; Dudek, Hanna M; Gómez Castellanos, J Rúben; Gutiérrez de Souza, Cora; Rovida, Stefano; Fraaije, Marco W; Mattevi, Andrea

2017-01-01

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these

Kynurenine 3-monooxygenase from Pseudomonas fluorescens: substrate-like inhibitors both stimulate flavin reduction and stabilize the flavin-peroxo intermediate yet result in the production of hydrogen peroxide.

Science.gov (United States)

Crozier-Reabe, Karen R; Phillips, Robert S; Moran, Graham R

2008-11-25

Kynurenine 3-monooxygenase (KMO) is a flavin-dependent hydroxylase that catalyzes the conversion of l-kynurenine (l-Kyn) to 3-hydroxykynurenine (3OHKyn) in the pathway for tryptophan catabolism. KMO inhibition has been widely suggested as an early treatment for stroke and other neurological disorders that involve ischemia. We have investigated the reductive and the oxidative half-reactions of a stable form of KMO from Pseudomonas fluorescens (KMO). The binding of l-Kyn by the enzyme is relatively slow and involves at least two reversible steps. The rate constant for reduction of the flavin cofactor by NADPH increases by a factor of approximately 2.5 x 10(3) when l-Kyn is bound. The rate of reduction of the KMO.l-Kyn complex is 160 s(-1), and the K(d) for the NADPH complex is 200 microM with charge-transfer absorption bands for the KMO(RED).l-Kyn.NADP(+) complex accumulating after reduction. The reduction potential of KMO is -188 mV and is unresponsive to the addition of l-Kyn or other inhibitory ligands. KMO inhibitors whose structures are reminiscent of l-Kyn such as m-nitrobenzoylalanine and benzoylalanine also stimulate reduction of flavin by NADPH and, in the presence of dioxygen, result in the stoichiometric liberation of hydrogen peroxide, diminishing the perceived therapeutic potential of inhibitors of this type. In the presence of the native substrate, the oxidative half-reaction exhibits triphasic absorbance data. A spectrum consistent with that of a peroxyflavin species accumulates and then decays to yield the oxidized enzyme. This species then undergoes minor spectral changes that, based on flavin difference spectra defined in the presence of 3OHKyn, can be correlated with product release. The oxidative half-reaction observed in the presence of saturating benzoylalanine or m-nitrobenzoylalanine also shows the accumulation of a peroxyflavin species that then decays to yield hydrogen peroxide without hydroxylation.

Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

Science.gov (United States)

Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

2016-08-01

N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more

The involvement of flavin-containing monooxygenase but not CYP3A4 in metabolism of itopride hydrochloride, a gastroprokinetic agent: comparison with cisapride and mosapride citrate.

Science.gov (United States)

Mushiroda, T; Douya, R; Takahara, E; Nagata, O

2000-10-01

The goals of the present study were to identify the enzyme responsible for metabolism of itopride hydrochloride (itopride) and to evaluate the likelihood of drug interaction involving itopride. In human liver microsomes, the involvement of flavin-containing monooxygenase in N-oxygenation, the major metabolic pathway of itopride, was indicated by the following results: inhibition by methimazole and thiourea, heat inactivation, and protection against heat inactivation by NADPH. When the effects of ketoconazole on the metabolism of itopride, cisapride, and mosapride citrate (mosapride) were examined using human liver microsomes, ketoconazole strongly inhibited the formation of the primary metabolites of cisapride and mosapride, but not itopride. Other cytochrome P450 (CYP) 3A4 inhibitors, cimetidine, erythromycin, and clarithromycin, also inhibited the metabolism of cisapride and mosapride. In an in vivo study, itopride (30 mg/kg), cisapride (1.5 mg/kg), or mosapride (3 mg/kg) was orally administered to male rats with or without oral pretreatment with ketoconazole (120 mg/kg) twice daily for 2 days. The ketoconazole pretreatment significantly increased the area under the serum concentration curve and the maximum serum concentration of cisapride and mosapride but had no significant effect on the pharmacokinetics of itopride. In addition, itopride did not inhibit five specific CYP-mediated reactions of human liver microsomes. These results suggest that itopride is unlikely to alter the pharmacokinetics of other concomitantly administered drugs.

Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

Science.gov (United States)

Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

2013-11-08

SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

International Nuclear Information System (INIS)

Eom, Hyun-Jeong; Ahn, Jeong-Min; Kim, Younghun; Choi, Jinhee

2013-01-01

In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO 3 in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO 3 did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO 3 . These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO 3 . • HIF-1 and PMK-1 were needed for AgNPs- and AgNO 3 -induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO 3 did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal amount of silver mass contained

Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

Energy Technology Data Exchange (ETDEWEB)

Eom, Hyun-Jeong; Ahn, Jeong-Min [School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743 (Korea, Republic of); Kim, Younghun [Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-dong, Nowon-gu, Seoul 139-701 (Korea, Republic of); Choi, Jinhee, E-mail: jinhchoi@uos.ac.kr [School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743 (Korea, Republic of)

2013-07-15

In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO{sub 3} in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO{sub 3} did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO{sub 3}. These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO{sub 3}. • HIF-1 and PMK-1 were needed for AgNPs- and AgNO{sub 3}-induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO{sub 3} did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal

Predicting the Metabolic Sites by Flavin-Containing Monooxygenase on Drug Molecules Using SVM Classification on Computed Quantum Mechanics and Circular Fingerprints Molecular Descriptors.

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Chien-Wei Fu

Full Text Available As an important enzyme in Phase I drug metabolism, the flavin-containing monooxygenase (FMO also metabolizes some xenobiotics with soft nucleophiles. The site of metabolism (SOM on a molecule is the site where the metabolic reaction is exerted by an enzyme. Accurate prediction of SOMs on drug molecules will assist the search for drug leads during the optimization process. Here, some quantum mechanics features such as the condensed Fukui function and attributes from circular fingerprints (called Molprint2D are computed and classified using the support vector machine (SVM for predicting some potential SOMs on a series of drugs that can be metabolized by FMO enzymes. The condensed Fukui function fA- representing the nucleophilicity of central atom A and the attributes from circular fingerprints accounting the influence of neighbors on the central atom. The total number of FMO substrates and non-substrates collected in the study is 85 and they are equally divided into the training and test sets with each carrying roughly the same number of potential SOMs. However, only N-oxidation and S-oxidation features were considered in the prediction since the available C-oxidation data was scarce. In the training process, the LibSVM package of WEKA package and the option of 10-fold cross validation are employed. The prediction performance on the test set evaluated by accuracy, Matthews correlation coefficient and area under ROC curve computed are 0.829, 0.659, and 0.877 respectively. This work reveals that the SVM model built can accurately predict the potential SOMs for drug molecules that are metabolizable by the FMO enzymes.

Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase.

Science.gov (United States)

Yi, Jicai; Liu, Lanna; Cao, Youpei; Li, Jiazuo; Mei, Mantong

2013-12-01

Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Shwing a Covalent Flavin-Aspartate Bond

International Nuclear Information System (INIS)

Podzelinska, K.; Latimer, R.; Bhattacharya, A.; Vining, L.; Zechel, D.; Jia, Z.

2010-01-01

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 (angstrom) resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

A Click Chemistry Approach towards Flavin-Cyclodextrin Conjugates-Bioinspired Sulfoxidation Catalysts

Czech Academy of Sciences Publication Activity Database

Tomanová, P.; Šturala, J.; Buděšínský, Miloš; Cibulka, R.

2015-01-01

Roč. 20, č. 11 (2015), s. 19837-19848 ISSN 1420-3049 Institutional support: RVO:61388963 Keywords : click chemistry * cyclodextrin * flavin * monooxygenase * oxidation * sulfoxides * green chemistry Subject RIV: CC - Organic Chemistry Impact factor: 2.465, year: 2015 http://www.mdpi.com/1420-3049/20/11/19667/htm

Flavin-dependent monooxygenases as a detoxification mechanism in insects: new insights from the arctiids (lepidoptera.

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Sven Sehlmeyer

2010-05-01

Full Text Available Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3. Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects

Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

Science.gov (United States)

Louie, Gordon; Noel, Joseph P.; Baran, Phil S.; Palfey, Bruce; Moore, Bradley S.

2013-01-01

Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization. PMID:24162851

[The effect of berberine administration of evaluation of the functional state of rat liver after ligation of common bile duct].

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Zverinskiĭ, I V; Mel'nichenko, N G; Poplavskiĭ, V A; Sut'ko, I P; Telegin, P G; Shliakhtun, A G

2013-01-01

On the eighth day after ligation of the common bile duct in rats a significant increase in the serum content of total lipids, cholesterol bilirubin and ALT, alkaline phosphatase, and gamma-glutamyltransferase was observed. In the microsomal fraction there was a marked decrease in the content and activity of microsomal monooxygenases. Introperitoneal injection of berberine (10 mg/kg) for 6 days caused a partial normalization of permeability of hepatocytes plasma membranes and activity microsomal flavin-containing monooxygenases. It is suggested that berberine is a substrate and inducer of flavin-containing monooxygenases. Membrane-stabilizing effect of berberine is probably realized at the level of inhibition of prooxidant status of liver cells.

Evolutionary recruitment of a flavin-dependent monooxygenase for stabilization of sequestered pyrrolizidine alkaloids in arctiids.

Science.gov (United States)

Langel, Dorothee; Ober, Dietrich

2011-09-01

Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis.

Science.gov (United States)

Masuda, Masaki; Freguia, Stefano; Wang, Yung-Fu; Tsujimura, Seiya; Kano, Kenji

2010-06-01

Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around -0.4V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results. Copyright 2009 Elsevier B.V. All rights reserved.

Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase.

Science.gov (United States)

Kim, Hyun Tae; Na, Byeong Kwan; Chung, Jiwoung; Kim, Sulhee; Kwon, Sool Ki; Cha, Hyunju; Son, Jonghyeon; Cho, Joong Myung; Hwang, Kwang Yeon

2018-04-19

Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

Science.gov (United States)

Chocklett, Samuel W; Sobrado, Pablo

2010-08-10

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism.

The 1.6 Å crystal structure of pyranose dehydrogenase from Agaricus meleagris rationalizes substrate specificity and reveals a flavin intermediate.

Directory of Open Access Journals (Sweden)

Tien Chye Tan

Full Text Available Pyranose dehydrogenases (PDHs are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O activates oxygen by a mechanism that proceeds via a covalent flavin C(4a-hydroperoxide intermediate. Although the flavin C(4a adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2

Formation of a Flavin-Linked Peptide

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Masayuki Morikawa

2014-07-01

Full Text Available In a previous study, we showed that formylmethylflavin (FMF can bind to cysteine. In this study, FMF was reacted with native peptides (CG and CKLVFF containing an N-terminal cysteine. The formation of flavin-CG and flavin-CKLVFF was confirmed using HPLC and ESI-MS. Storage of flavin-CKLVFF in DMSO at −30 °C for 7 days resulted in no detectable deposition. In contrast, flavin-CKLVFF formed deposits when stored in water at −30 °C for 1 day, but no deposit was observed in the aqueous solution of flavin-CKLVFF after 7 days storage in the presence of 0.1% Triton X-100.

Structural basis of kynurenine 3-monooxygenase inhibition.

Science.gov (United States)

Amaral, Marta; Levy, Colin; Heyes, Derren J; Lafite, Pierre; Outeiro, Tiago F; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S

2013-04-18

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer's disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases.

Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

Science.gov (United States)

Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

2010-12-01

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase

Science.gov (United States)

Schlömann, Michael; van Berkel, Willem J.H.; Gassner, George T.

2013-01-01

StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst. PMID:24157359

Structure and function of para-hydroxybenzoate hydroxylase

NARCIS (Netherlands)

Bolt, van der F.J.T.

1999-01-01

Enzymes which utilize molecular oxygen to either hydroxylate or cleave an aromatic ring are known as monooxygenases and dioxygenases, respectively. These enzymes contain a non-protein group such as heme, flavin, pterin or a transition metal ion in their active site, for oxygen activation.

[Effects of berberine on the recovery of rat liver xenobiotic-metabolizing enzymes after partial hepatectomy].

Science.gov (United States)

Zverinsky, I V; Zverinskaya, H G; Sutsko, I P; Telegin, P G; Shlyahtun, A G

2015-01-01

We have studied the effect of berberine on the recovery processes of liver xenobiotic-metabolizing function during its compensatory growth after 70% partial hepatectomy. It was found the hepatic ability to metabolize foreign substances are not restored up to day 8. Administration of berberine (10 mg/kg intraperitoneally) for 6 days led to normalization of both cytochrome P450-dependent and flavin-containing monooxygenases. It is suggested that in the biotransformation of berberine involved not only cytochrome P450, but also flavin-containing monooxygenases.

YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana

KAUST Repository

Kim, Jeong Im; Murphy, Angus S.; Baek, Dongwon; Lee, Shin-Woo; Yun, Dae-Jin; Bressan, Ray A.; Narasimhan, Meena L.

2011-01-01

The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical

N-terminus determines activity and specificity of styrene monooxygenase reductases.

Science.gov (United States)

Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

2017-12-01

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.

Science.gov (United States)

Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki

2013-10-01

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Flavin-N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme.

Science.gov (United States)

Dai, Yumin; Kizjakina, Karina; Campbell, Ashley C; Korasick, David A; Tanner, John J; Sobrado, Pablo

2018-01-04

The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2-HAH was shown to be monomeric in solution and contained a non-covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox-neutral, 2-HAH was active only in the reduced state. A covalent flavin-substrate intermediate, consistent with the flavin-acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small-angle X-ray scattering was consistent with 2-HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2-HAH as a novel noncanonical flavoenzyme. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase

Science.gov (United States)

Hutchinson, Jonathan P.; Rowland, Paul; Taylor, Mark R. D.; Christodoulou, Erica M.; Haslam, Carl; Hobbs, Clare I.; Holmes, Duncan S.; Homes, Paul; Liddle, John; Mole, Damian J.; Uings, Iain; Walker, Ann L.; Webster, Scott P.; Mowat, Christopher G.; Chung, Chun-Wa

2017-06-01

Kynurenine-3-monooxygenase (KMO) is a key FAD-dependent enzyme of tryptophan metabolism. In animal models, KMO inhibition has shown benefit in neurodegenerative diseases such as Huntington's and Alzheimer's. Most recently it has been identified as a target for acute pancreatitis multiple organ dysfunction syndrome (AP-MODS); a devastating inflammatory condition with a mortality rate in excess of 20%. Here we report and dissect the molecular mechanism of action of three classes of KMO inhibitors with differentiated binding modes and kinetics. Two novel inhibitor classes trap the catalytic flavin in a previously unobserved tilting conformation. This correlates with picomolar affinities, increased residence times and an absence of the peroxide production seen with previous substrate site inhibitors. These structural and mechanistic insights culminated in GSK065(C1) and GSK366(C2), molecules suitable for preclinical evaluation. Moreover, revising the repertoire of flavin dynamics in this enzyme class offers exciting new opportunities for inhibitor design.

Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

DEFF Research Database (Denmark)

Paspaliari, Dafni Katerina; Loose, Jennifer S. M.; Larsen, Marianne Halberg

2015-01-01

Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and ChiB) and a ......Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and Chi...... but different product profiles depending on the substrate. In LPMO-chitinase synergy experiments, CBP21 is able to boost the activity of both ChiA and ChiB more than LmLPMO10. Product analysis of the synergy assays revealed that the chitinases were unable to efficiently hydrolyse the LPMO products...... (chitooligosaccharide aldonic acids) with a degree of polymerization below four (ChiA and SmChiC) or three (ChiB). Gene transcription and protein expression analysis showed that LmLPMO10 is neither highly transcribed, nor abundantly secreted during the growth of L. monocytogenes in a chitin-containing medium...

Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry

DEFF Research Database (Denmark)

Westereng, Bjørge; Arntzen, Magnus Ø.; Wittrup Agger, Jane

2017-01-01

Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the num...

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

Science.gov (United States)

White, Mark D.; Payne, Karl A.P.; Fisher, Karl; Marshall, Stephen A.; Parker, David; Rattray, Nicholas J.W.; Trivedi, Drupad K.; Goodacre, Royston; Rigby, Stephen E.J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

2016-01-01

Ubiquinone, or coenzyme Q, is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains1. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor2. Despite structural and biochemical characterization of UbiX as an FMN-binding protein, no decarboxylase activity has been detected3–4. We report here that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD5. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases6–7, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyl transferase mechanism of UbiX resembles that of the terpene synthases8. The active site environment is dominated by π-systems, which assist phosphate-C1’ bond breakage following FMN reduction, leading to formation of the N5-C1’ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3’-C6 bond. The study establishes the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoire. PMID:26083743

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

Science.gov (United States)

Menon, Binuraj R K; Latham, Jonathan; Dunstan, Mark S; Brandenburger, Eileen; Klemstein, Ulrike; Leys, David; Karthikeyan, Chinnan; Greaney, Michael F; Shepherd, Sarah A; Micklefield, Jason

2016-10-04

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli

International Nuclear Information System (INIS)

Gao, Benlian; Bertrand, Adam; Boles, William H.; Ellis, Holly R.; Mallett, T. Conn

2005-01-01

Crystallization of the native and SeMet FMN reductase protein of the E. coli alkanesulfonate monooxygenase two-component enzyme system is reported. The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 Å resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin

Synthesis and Characterization of Naphthalenediimide-Functionalized Flavin Derivatives

Directory of Open Access Journals (Sweden)

Graeme Cooke

2013-04-01

Full Text Available Two acceptor–acceptor dyads have been synthesized featuring a flavin moiety and a naphthalenediimide (NDI unit. The NDI unit is linked to the flavin through a short spacer group via either the N(3 or N(10 positions of the flavin. We have investigated the UV-Vis and redox properties of these multi-electron accepting systems which indicate that these materials display the collective properties of their component systems. Fluorescence spectroscopy measurements have revealed that their emission properties are dominated by the flavin unit.

Daily fluctuation of hepatic P450 monooxygenase activities in male rats is controlled by the suprachiasmatic nucleus but remains unaffected by adrenal hormones.

Science.gov (United States)

Furukawa, T; Manabe, S; Watanabe, T; Sehata, S; Sharyo, S; Okada, T; Mori, Y

1999-09-01

Hepatic P450 monooxygenase activities, which strongly influence the efficacy and/or toxicity of drugs, are known to fluctuate daily. We also know that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities fluctuate daily, with apparently high values during the dark period in male rats. However, there is little knowledge about the factors that regulate daily fluctuation of P450 monooxygenase activities. In the present study using rats, we induced lesions in the suprachiasmatic nucleus (SCN) of the brain, the known site of the body's internal clock, and examined the effects on the daily fluctuation of the ACD activities to clarify the relationship between the SCN and the daily fluctuation of P450 monooxygenase activities. In addition, adrenalectomy was performed to re-evaluate the influence of adrenal hormones on the P450 activities. Our results indicated that daily fluctuations of the hepatic ACD activities were completely eliminated in the SCN-lesioned rats. However, the ACD activities in the adrenalectomized rats showed apparent daily fluctuations with high values during the dark period and low values during the light period. Therefore, this study demonstrated that the daily fluctuation of the hepatic P450 monooxygenase activities in male rats is controlled by the SCN but remains unaffected by the adrenal hormones.

Secretion of Flavins by Three Species of Methanotrophic Bacteria▿ †

OpenAIRE

Balasubramanian, Ramakrishnan; Levinson, Benjamin T.; Rosenzweig, Amy C.

2010-01-01

We detected flavins in the growth medium of the methanotrophic bacterium Methylocystis species strain M. Flavin secretion correlates with growth stage and increases under iron starvation conditions. Two other methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), secrete flavins, suggesting that flavin secretion may be common to many methanotrophic bacteria.

Regulation of cytochrome P-450 monooxygenases in the mouse

International Nuclear Information System (INIS)

Kelley, M.F.

1986-01-01

Recently, the compound 1,4-bis[2-(3,4-dichloropyridyloxy)] benzene (TCPOBOP) has been identified as a highly potent phenobabital-like agonist in mice. This finding has led to the suggestion that a receptor-mediated process may govern the induction of cytochrome P-450 monooxygenases by phenobarbital and phenobarbital-like agonists. This dissertation examines: (1) the effects of structural alterations of the TCPOBOP molecule on enzyme induction activity, (2) the induction response to phenobarbital and TCPOBOP among inbred mouse strains, (3) the spectrum of monooxygenase activities induced by phenobarbital and TCPOBOP compared to 3-methylcholanthrene, isosafrole and pregnenolone 16α-carbonitrile (PCN) and (4) the binding of [ 3 H] TCPOBOP in hepatic cytosol. Changes in the structure of the pyridyloxy or benzene rings markedly affect enzyme induction activity and provide additional indirect evidence for a receptor-mediated response. An evaluation of monooxygenase induction by TCPOBOP for 27 inbred mouse strains and by phenobarbital for 15 inbred mouse strains failed to identify a strain which was completely nonresponsive to these compounds, although several strains exhibited decreased responsiveness for select monooxygenase reactions. TCPOBOP, PCN and phenobarbital were all found to significantly increase the rate of hydroxylation of testosterone at the 2α-, 6β- and 15β- positions but only TCPOBOP and phenobarbital dramatically increased the rate of pentoxyresorufin O-dealkylation. The results demonstrates that TCPOBOP most closely resembles phenobarbital in its mode of monooxygenase induction in mice. Sucrose density gradient analysis of [ 3 H] TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of [ 3 H] TCPOBOP to cytosolic marcomolecular elements

Genotyping of flavin-containing mono-oxygenase 3 (FMO3) gene by ...

African Journals Online (AJOL)

63.40%) of the 306 samples were genotyped using MAMA-PCR and 42 (13.72%) of the 306 samples were genotyped by both of PCR-RFLP and MAMA-PCR and genotyping data were validated by DNA sequencing. The results show that the ...

Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6.

Directory of Open Access Journals (Sweden)

Alexandre Ismail

2016-01-01

Full Text Available Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6, a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations or completely (a G248R-L382E double-mutation blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.

Lyophilization conditions for the storage of monooxygenases

NARCIS (Netherlands)

van Beek, Hugo L.; Beyer, Nina; Janssen, Dick B.; Fraaije, Marco W.

2015-01-01

Cyclohexanone monooxygenase (CHMO) was used as a model enzyme to find suitable freeze-drying conditions for long-term storage of an isolated monooxygenase. CHMO is a Baeyer-Villiger monooxygenase (BVMO) known for its ability to catalyze a large number of oxidation reactions. With a focus on

Cytochrome P450 monooxygenases and insecticide resistance in insects.

OpenAIRE

Bergé, J B; Feyereisen, R; Amichot, M

1998-01-01

Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

Science.gov (United States)

Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

2014-01-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

Exploiting the enantioselectivity of Baeyer-Villiger monooxygenases via boron oxidation

NARCIS (Netherlands)

Brondani, Patricia B.; Dudek, Hanna; Reis, Joel S.; Fraaije, Marco W.; Andrade, Leandro H.

2012-01-01

The enantioselective carbon-boron bond oxidation of several chiral boron-containing compounds by Baeyer-Villiger monooxygenases was evaluated. PAMO and M446G PAMO conveniently oxidized 1-phenylethyl boronate into the corresponding 1-(phenyl)ethanol (ee = 82-91%). Cyclopropyl boronic esters were also

The effect of flavin electron shuttles in microbial fuel cells current production

Energy Technology Data Exchange (ETDEWEB)

Velasquez-Orta, Sharon B. [Newcastle Univ., Newcastle upon Tyne (United Kingdom). School of Civil Engineering and Geosciences; Newcastle Univ., Newcastle upon Tyne (United Kingdom). School of Chemical Engineering and Advanced Materials; Head, Ian M.; Curtis, Thomas P. [Newcastle Univ., Newcastle upon Tyne (United Kingdom). School of Civil Engineering and Geosciences; Scott, Keith [Newcastle Univ., Newcastle upon Tyne (United Kingdom). School of Chemical Engineering and Advanced Materials; Lloyd, Jonathan R.; Canstein, Harald von [Manchester Univ. (United Kingdom). School of Earth, Atmospheric and Environmental Sciences

2010-02-15

The effect of electron shuttles on electron transfer to microbial fuel cell (MFC) anodes was studied in systems where direct contact with the anode was precluded. MFCs were inoculated with Shewanella cells, and flavins used as the electron shuttling compound. In MFCs with no added electron shuttles, flavin concentrations monitored in the MFCs' bulk liquid increased continuously with FMN as the predominant flavin. The maximum concentrations were 0.6 {mu}M for flavin mononucleotide and 0.2 {mu}M for riboflavin. In MFCs with added flavins, micro-molar concentrations were shown to increase current and power output. The peak current was at least four times higher in MFCs with high concentrations of flavins (4.5-5.5 {mu}M) than in MFCs with low concentrations (0.2-0.6 {mu}M). Although high power outputs (around 150 mW/m{sup 2}) were achieved in MFCs with high concentrations of flavins, a Clostridium-like bacterium along with other reactor limitations affected overall coulombic efficiencies (CE) obtained, achieving a maximum CE of 13%. Electron shuttle compounds (flavins) permitted bacteria to utilise a remote electron acceptor (anode) that was not accessible to the cells allowing current production until the electron donor (lactate) was consumed. (orig.)

Time-resolved fluorescence analysis of the mobile flavin cofactor

Indian Academy of Sciences (India)

Conformational heterogeneity of the FAD cofactor in -hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations ...

Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

Science.gov (United States)

Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

2017-07-01

How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

Interaction of ZnS nanoparticles with flavins and glucose oxidase: A fluorimetric investigation

International Nuclear Information System (INIS)

Chatterjee, Anindita; Priyam, Amiya; Ghosh, Debasmita; Mondal, Somrita; Bhattacharya, Subhash C.; Saha, Abhijit

2012-01-01

Interactions of luminescence, water soluble ZnS nanoparticles (NPs) with flavins and glucose oxidase have been thoroughly investigated through optical spectroscopy. The photoluminescence of ZnS nanoparticles was quenched severely (∼60%) by riboflavin while other flavins such as flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) show quenching to different extents under analogous conditions. However, interestingly no effect in luminescence intensity of ZnS NPs was observed with protein bound flavins such as in glucose oxidase. Fluorescence lifetime measurement confirmed the quenching to be static in nature. Scavenging of photo-generated electron of ZnS nanoparticles by the flavin molecules may be attributed to the decrease in luminescence intensity. Quenching of ZnS nanoparticles with flavins follows the linear Stern–Volmer plot. The Stern–Volmer constants decreased in the following order: K S−V (Riboflavin)> K S−V (FAD)> K S−V (FMN). This interaction study could generate useful protocol for the fluorimetric determination of riboflavin (vitamin B 2 ) content and also riboflavin status in biological systems. - Highlights: ► Unique interaction specificity of ZnS nanoparticles with flavins has been explored. ► Unlike protein-bound flavin, fluorescence of free flavins was quenched by ZnS nanoparticles. ► FMN and FAD show quenching to different extents under analogous conditions. ► Fluorescence lifetime measurement confirmed the quenching to be static in nature. ► This study is useful for probing riboflavin in biological systems.

A biomimetic redox flow battery based on flavin mononucleotide.

Science.gov (United States)

Orita, Akihiro; Verde, Michael G; Sakai, Masanori; Meng, Ying Shirley

2016-10-21

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.

A biomimetic redox flow battery based on flavin mononucleotide

Science.gov (United States)

Orita, Akihiro; Verde, Michael G.; Sakai, Masanori; Meng, Ying Shirley

2016-10-01

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

Science.gov (United States)

Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

2014-03-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase.

Science.gov (United States)

Bauman, Andrew T; Broers, Brenda A; Kline, Chelsey D; Blackburn, Ninian J

2011-12-20

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase

Biocatalysts for selective introduction of oxygen

DEFF Research Database (Denmark)

Leak, D. J.; Sheldon, R. A.; Woodley, John

2009-01-01

Three types of oxygenase biocatalysts are treated in detail in this review: the non-haem iron alkene mono-oxygenases, the haem and vanadium haloperoxidases, and flavin-based Baeyer-Villiger mono-oxygenases. Other oxygenases are briefly included for comparison. Characteristics of the biocatalysts ...

A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

DEFF Research Database (Denmark)

Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

2017-01-01

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty......-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used...... as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes...

Interaction of ZnS nanoparticles with flavins and glucose oxidase: A fluorimetric investigation

Energy Technology Data Exchange (ETDEWEB)

Chatterjee, Anindita; Priyam, Amiya; Ghosh, Debasmita; Mondal, Somrita [UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8, Bidhannagar, Kolkata 700098 (India); Bhattacharya, Subhash C. [Department of Chemistry, Jadavpur University, Kolkata 700032 (India); Saha, Abhijit, E-mail: abhijit@alpha.iuc.res.in [UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8, Bidhannagar, Kolkata 700098 (India)

2012-03-15

Interactions of luminescence, water soluble ZnS nanoparticles (NPs) with flavins and glucose oxidase have been thoroughly investigated through optical spectroscopy. The photoluminescence of ZnS nanoparticles was quenched severely ({approx}60%) by riboflavin while other flavins such as flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) show quenching to different extents under analogous conditions. However, interestingly no effect in luminescence intensity of ZnS NPs was observed with protein bound flavins such as in glucose oxidase. Fluorescence lifetime measurement confirmed the quenching to be static in nature. Scavenging of photo-generated electron of ZnS nanoparticles by the flavin molecules may be attributed to the decrease in luminescence intensity. Quenching of ZnS nanoparticles with flavins follows the linear Stern-Volmer plot. The Stern-Volmer constants decreased in the following order: K{sub S-V} (Riboflavin)> K{sub S-V} (FAD)> K{sub S-V} (FMN). This interaction study could generate useful protocol for the fluorimetric determination of riboflavin (vitamin B{sub 2}) content and also riboflavin status in biological systems. - Highlights: Black-Right-Pointing-Pointer Unique interaction specificity of ZnS nanoparticles with flavins has been explored. Black-Right-Pointing-Pointer Unlike protein-bound flavin, fluorescence of free flavins was quenched by ZnS nanoparticles. Black-Right-Pointing-Pointer FMN and FAD show quenching to different extents under analogous conditions. Black-Right-Pointing-Pointer Fluorescence lifetime measurement confirmed the quenching to be static in nature. Black-Right-Pointing-Pointer This study is useful for probing riboflavin in biological systems.

Monooxygenase activitity in Aedes aegypti population in Tembalang subdistrict, Semarang city

Directory of Open Access Journals (Sweden)

Dyah Widiastuti

2015-06-01

Full Text Available Dengue Haemorrhagic Fever (DHF is a major health problem in Tembalang sub district, Semarang City. Fogging with insecticide applications was done frequently as an effort to control Dengue vectors. The use of insecticides from the same class in a long time can lead to resistance in mosquitos’ population. The research aimed to observe the activity of monooxygenases in Aedes aegypti populations in Tembalang Subdistrict, Semarang. The study was conducted during February-November 2014 with a cross-sectional design in 10 villages in Tembalang Subdistirict, Semarang City. Field strains of Ae. aegypti eggs were collected using ovitraps. The collected eggs were grown under standard condition to adult mosquitoes. Mosquitos’ homogenate were stored at -85C and used for biochemical assays. The results showed there was increased monooxygenases activity in Ae. aegypti populations. Resistance to synthetic pyrethroid insecticide in Ae. aegypti mosquitoes population in Tembalang Subdistrict might be caused by the mechanism of detoxification enzymes in particular monooxygenases

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

Energy Technology Data Exchange (ETDEWEB)

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.

2016-01-29

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH₃) to nitrite (NO₂^₋) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH₄⁺-dependent O₂uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and

Flavin-catalyzed redox tailoring reactions in natural product biosynthesis.

Science.gov (United States)

Teufel, Robin

2017-10-15

Natural products are distinct and often highly complex organic molecules that constitute not only an important drug source, but have also pushed the field of organic chemistry by providing intricate targets for total synthesis. How the astonishing structural diversity of natural products is enzymatically generated in biosynthetic pathways remains a challenging research area, which requires detailed and sophisticated approaches to elucidate the underlying catalytic mechanisms. Commonly, the diversification of precursor molecules into distinct natural products relies on the action of pathway-specific tailoring enzymes that catalyze, e.g., acylations, glycosylations, or redox reactions. This review highlights a selection of tailoring enzymes that employ riboflavin (vitamin B2)-derived cofactors (FAD and FMN) to facilitate unusual redox catalysis and steer the formation of complex natural product pharmacophores. Remarkably, several such recently reported flavin-dependent tailoring enzymes expand the classical paradigms of flavin biochemistry leading, e.g., to the discovery of the flavin-N5-oxide - a novel flavin redox state and oxygenating species. Copyright © 2017 Elsevier Inc. All rights reserved.

Absorption and emission spectroscopic characterisation of a pyrene-flavin dyad

International Nuclear Information System (INIS)

Shirdel, J.; Penzkofer, A.; Prochazka, R.; Shen, Z.; Strauss, J.; Daub, J.

2007-01-01

The pyrene-flavin (isoalloxazine) dyad, PFD {C 44 H 31 N 5 O 5 ; CA Index name: 1-pyrenepropanoic acid, α-[[4,10-dihydro-2,4-dioxo-10- phenylbenzo[g]pteridin-3(2H)-yl)acetyl]amino]-, phenylmethyl ester (αR)-(9Cl); CA Registry number: 618907-57-6}, dissolved in either dichloromethane or acetonitrile is characterized by absorption and emission spectroscopy. Absorption cross-section spectra, stimulated emission cross-section spectra, fluorescence quantum distributions, quantum yields, and degrees of fluorescence polarisation are determined. The fluorescence decay after femtosecond pulse excitation is determined by fluorescence up-conversion. The ground-state absorption recovery is determined by picosecond pump and probe transmission measurements. The dye photo-stability is investigated by observation of absorption spectral changes due to prolonged blue-light excitation. The absorption spectrum of PFD dyad resembles the superposition of the absorption of isoalloxazine (flavin) and 1-methylpyrene. Long-wavelength photo-excitation of the flavin moiety causes fluorescence quenching by ground-state electron transfer from pyrene to isoalloxazine. Short-wavelength photo-excitation of the pyrene moiety causes (i) excited-state electron transfer from pyrene to isoalloxazine, and (ii) Foerster-type energy transfer from pyrene to flavin followed by ground-state electron transfer from pyrene to flavin.

Identification of the intermediates of in vivo oxidation of 1 ,4-dioxane by monooxygenase-containing bacteria.

Science.gov (United States)

Mahendra, Shaily; Petzold, Christopher J; Baidoo, Edward E; Keasling, Jay D; Alvarez-Cohen, Lisa

2007-11-01

1,4-dioxane is a probable human carcinogen and an emerging water contaminant. Monooxygenase-expressing bacteria have been shown to degrade dioxane via growth-supporting as well as cometabolic mechanisms. In this study, the intermediates of dioxane degradation by monooxygenase-expressing bacteria were determined by triple quadrupole-mass spectrometry and Fourier transform ion cyclotron resonance-mass spectrometry. The major intermediates were identified as 2-hydroxyethoxyacetic acid (HEAA), ethylene glycol, glycolate, and oxalate. Studies with uniformly labeled 14C dioxane showed that over 50% of the dioxane was mineralized to CO2 by CB1190, while 5% became biomass-associated after 48 h. Volatile organic acids and non-volatiles, respectively, accounted for 20 and 11% of the radiolabeled carbon. Although strains cometabolizing dioxane exhibited limited transformation capacities, nearly half of the initial dioxane was recovered as CO2. On the basis of these analytical results, we propose a pathway for dioxane oxidation by monooxygenase-expressing cells in which dioxane is first converted to 2-hydroxy-1,4-dioxane, which is spontaneously oxidized to HEAA. During a second monooxygenation step, HEAA is further hydroxylated, resulting in a mixture of dihydroxyethoxyacetic acids with a hydroxyl group at the ortho or para position. After cleavage of the second ether bond, small organic molecules such as ethylene glycol, glycolate, glyoxalate, and oxalate are progressively formed, which are then mineralized to CO2 via common cellular metabolic pathways. Bioremediation of dioxane via this pathway is not expected to cause an accumulation of toxic compounds in the environment.

Extracellular Electron Transfer Mediated by Flavins in Gram-positive Bacillus sp. WS-XY1 and Yeast Pichia stipitis

International Nuclear Information System (INIS)

Wu, Song; Xiao, Yong; Wang, Lu; Zheng, Yue; Chang, Kenlin; Zheng, Zhiyong; Yang, Zhaohui; Varcoe, John R.; Zhao, Feng

2014-01-01

Extracellular electron transfer (EET) of microorganisms represents a communicative bridge between the interior and exterior of the cells. Most prior EET studies have focused on Gram-negative bacteria. However, fungi and Gram-positive bacteria, that contain dense cellular walls, have rarely been reported. Herein, two model dense cell wall microorganisms (Bacillus sp. WS-XY1 and the yeast Pichia stipitis) were identified to be electrochemically active. Further analysis indicated that the two microorganisms were able to secrete flavins to mediate their EET. The discovery, that dense cell wall containing microorganisms can undertake mediated EET, adds to the body of knowledge towards building a comprehensive understanding of biogeochemical and bioelectrical processes

Effect of flavin compounds on uranium(VI) reduction- kinetic study using electrochemical methods with UV-vis spectroscopy

International Nuclear Information System (INIS)

Yamasaki, Shinya; Tanaka, Kazuya; Kozai, Naofumi; Ohnuki, Toshihiko

2017-01-01

The reduction of uranium hexavalent (U(VI)) to tetravalent (U(IV)) is an important reaction because of the change in its mobility in the natural environment. Although the flavin mononucleotide (FMN) has acted as an electron shuttle for the U(VI) reduction in vivo system, which is called an electron mediator, only the rate constant for the electron transfer from FMN to U(VI) has been determined. This study examined the rate constant for the U(VI) reduction process by three flavin analogues (riboflavin, flavin mononucleotide, flavin adenine dinucleotide) to elucidate their substituent group effect on the U(VI) reduction rate by electrochemical methods. The formation of the U(IV) was monitored by UV-vis spectrometry at 660 nm during the constant potential electrolysis of the U(VI) solution in the presence of the mediator. The cyclic voltammograms indicated that the three flavin analogues behaved as electron mediator to reduce U(VI). The logarithmic rate constant for the U(VI) reduction was related to the standard redox potential of the mediators. This linear relationship indicated that the redox-active group of the mediator and the substituent group of the mediator dominate capability of the U(VI) reduction and its rate, respectively. The apparent reduction potential of U(VI) increased about 0.2 V in the presence of the mediators, which strongly suggests that the biological electron mediator makes the U(VI) reduction possible even under more oxidative conditions. - Highlights: • The rate constant for the U(VI) reduction by flavin analogues was determined. • The flavins showed a mediator effect on the U(VI) reduction. • The logarithmic rate constants for the U(VI) reduction was proportional to redox potential of the mediator. • The presence of the mediator increased about 0.2 V apparent redox potential of U(VI) to U(IV).

Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida.

Science.gov (United States)

Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

2008-04-01

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.

IMMOBILIZATION OF FLAVIN ON HIGHLY POROUS POLYMERIC DISKS - 3 ROUTES TO A CATALYTICALLY ACTIVE MEMBRANE

NARCIS (Netherlands)

SCHOO, HFM; CHALLA, G; ROWATT, B; SHERRINGTON, DC

Disks obtained by polymerization of high internal phase emulsions (Polyhipe) had completely open pore structures and were used as a carrier material for the immobilisation of 10-ethyl-isoalloxazine ('flavin'). Three methods for immobilization are described: (1) direct modification of

Terbinafine Resistance Mediated by Salicylate 1-Monooxygenase in Aspergillus nidulans

Science.gov (United States)

Graminha, Marcia A. S.; Rocha, Eleusa M. F.; Prade, Rolf A.; Martinez-Rossi, Nilce M.

2004-01-01

Resistance to antifungal agents is a recurring and growing problem among patients with systemic fungal infections. UV-induced Aspergillus nidulans mutants resistant to terbinafine have been identified, and we report here the characterization of one such gene. A sib-selected, 6.6-kb genomic DNA fragment encodes a salicylate 1-monooxygenase (salA), and a fatty acid synthase subunit (fasC) confers terbinafine resistance upon transformation of a sensitive strain. Subfragments carrying salA but not fasC confer terbinafine resistance. salA is present as a single-copy gene on chromosome VI and encodes a protein of 473 amino acids that is homologous to salicylate 1-monooxygenase, a well-characterized naphthalene-degrading enzyme in bacteria. salA transcript accumulation analysis showed terbinafine-dependent induction in the wild type and the UV-induced mutant Terb7, as well as overexpression in a strain containing the salA subgenomic DNA fragment, probably due to the multicopy effect caused by the transformation event. Additional naphthalene degradation enzyme-coding genes are present in fungal genomes, suggesting that resistance could follow degradation of the naphthalene ring contained in terbinafine. PMID:15328121

A biomimetic redox flow battery based on flavin mononucleotide

OpenAIRE

Orita, A; Verde, MG; Sakai, M; Meng, YS

2016-01-01

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactio...

Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus.

Science.gov (United States)

Ul-Hasan, Sabah; Burgess, Daniel M; Gajewiak, Joanna; Li, Qing; Hu, Hao; Yandell, Mark; Olivera, Baldomero M; Bandyopadhyay, Pradip K

2013-11-01

Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of Flavin7 on allergen induced hyperreactivity of airways

Directory of Open Access Journals (Sweden)

Franova S

2009-12-01

Full Text Available Abstract Some studies have suggested that the polyphenolic compounds might reduce the occurrence of asthma symptoms. The aim of our experiments was to evaluate the effects of 21 days of the flavonoid Flavin7 administration on experimentally induced airway inflammation in ovalbumin-sensitized guinea pigs. We assessed tracheal smooth muscle reactivity by an in vitro muscle-strip method; changes in airway resistance by an in vivo plethysmographic method; histological picture of tracheal tissue; and the levels of interleukin 4 (IL-4, and interleukin 5 (IL-5 in bronchoalveolar lavage fluid (BALF. Histological investigation of tracheal tissue and the concentrations of the inflammatory cytokines IL-4 and IL-5 in BALF were used as indices of airway inflammation. Administration of Flavin7 caused a significant decrease of specific airway resistance after histamine nebulization and a decline in tracheal smooth muscle contraction amplitude in response to bronchoconstricting mediators. Flavin7 minimized the degree of inflammation estimated on the basis of eosinophil calculation and IL-4 and IL-5 concentrations. In conclusion, administration of Flavin7 showed bronchodilating and anti-inflammatory effects on allergen-induced airway inflammation.

Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

Science.gov (United States)

Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana

2016-01-01

Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms. © 2015 CSIC New Phytologist © 2015 New Phytologist Trust.

The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

Science.gov (United States)

Park, Seon-Ha; Kang, Ji-Yeon; Kim, Dong-Hyun; Ahn, Taeho; Yun, Chul-Ho

2012-11-01

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 μM for MTT and kcat =6580 min(-1), Km =51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

Science.gov (United States)

van der Werf, Mariët J.; Swarts, Henk J.; de Bont, Jan A. M.

1999-01-01

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In

How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

Directory of Open Access Journals (Sweden)

Daniel eNohr

2015-09-01

Full Text Available Electron paramagnetic resonance (EPR spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage, and BLUF (blue-light using FAD domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

Science.gov (United States)

Nohr, Daniel; Rodriguez, Ryan; Weber, Stefan; Schleicher, Erik

2015-01-01

Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells.

Science.gov (United States)

Yang, Ming-Yeh; Chang, Chih-Jui; Chen, Liang-Yü

2017-08-01

Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study. Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462nm). Under blue light irradiation at 3.744J/cm 2 (=0.52mW/cm 2 irradiated for 2h), the minimal lethal dose is 3.125μM and the median lethal doses (LD 50 ) for FMN and FAD are 6.5μM and 7.2μM, respectively. Individual exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic photosensitizer for cancer research. Copyright © 2017 Elsevier B.V. All rights reserved.

A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.

Science.gov (United States)

Pierce, Brian C; Agger, Jane Wittrup; Zhang, Zhenghong; Wichmann, Jesper; Meyer, Anne S

2017-09-08

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose - a greater than 75% increase over the same treatment without LPMO addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

International Nuclear Information System (INIS)

Distefano, M.D.; Au, K.G.; Walsh, C.T.

1989-01-01

Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys 135 Cys 140 , catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys 135 (Ala 135 Cys 140 ), Cys 14 (Cys 135 Ala 140 ), or both (Ala 135 Ala 140 ). Additionally, they have made double mutants that lack Cys 135 (Ala 135 Cys 139 Cys 140 ) or Cys 140 (Cys 135 Cys 139 Ala 140 ) but introduce a new Cys in place of Gly 139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH 2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala 135 Cys 139 Cys 14 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala 135 Cys 140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys 135 and Cys 140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate

Photophysical properties of novel Porphyrin-Flavin Dyads

International Nuclear Information System (INIS)

Stark, S.

2001-10-01

Photosynthesis belongs to the fundamentals of life on earth, therefore it is an important matter in natural sciences. The basic principle of photosynthesis is the transformation of solar light into chemical energy. The starting steps of photosynthesis are light-induced energy- and electron-transfer-steps with singular efficiency. One attempt to enlighten the molecular processes involved is to synthesize simpler model systems with similar properties. Important research goals are the dependencies of light-induced processes on distance and orientation of donor and acceptor. A second aim next to the clarification of the molecular conditions of photosynthesis is to create molecular light-driven machines. The most simple so-called biomimetic model system consists of an electron-donor connected to an electron-acceptor via a spacer-group. This simplest form is also referred to as dyad. Beyond dyads far more complicated compounds have been introduced consisting of several donors and/or acceptors, so-called triads, tetrads, pentads etc. Usually porphyrin serves as electron-donor. Next to chinones several other electron-acceptors are used, e.g. anthracene, pyromellitimide and fullerene. Artificial photosynthetic centers are often more stable and/or the excited states are easier to detect compared to the natural photosynthetic center. The photophysical characteristics of four dyads are reported in this work. The dyads consist of porphyrin (either free-base or zinc-metallated) and flavin, connected by different spacers. These dyads reveal photo-induced electron transfer from porphyrin to flavin and energy-transfer in the reversed direction with different efficiencies. The object of the study is the dependency of these processes on the structural features. The spacer of the dyads 1a-1c is an aromatic bridge which leads to well defined donor-acceptor distances. Because of this structure conjugation through the spacer is increased, whereas the absorption in the visible and near UV

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

Science.gov (United States)

Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O

2009-07-07

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

A novel methanotroph in the genus Methylomonas that contains a distinct clade of soluble methane monooxygenase.

Science.gov (United States)

Nguyen, Ngoc-Loi; Yu, Woon-Jong; Yang, Hye-Young; Kim, Jong-Geol; Jung, Man-Young; Park, Soo-Je; Roh, Seong-Woon; Rhee, Sung-Keun

2017-10-01

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2-97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.

Reconstitution of β-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

International Nuclear Information System (INIS)

Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.

2006-01-01

CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards β-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 o C. In this system, β-carotene was hydroxylated to β-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low β-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated β-carotene at 3- and also 3'-positions, resulting in β-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol β-cryptoxanthin produced per nmol P450 per min

Sub-millitesla magnetic field effects on the recombination reaction of flavin and ascorbic acid radicals

Energy Technology Data Exchange (ETDEWEB)

Evans, Emrys W.; Henbest, Kevin B.; Timmel, Christiane R., E-mail: christiane.timmel@chem.ox.ac.uk, E-mail: stuart.mackenzie@chem.ox.ac.uk [Department of Chemistry, Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford (United Kingdom); Kattnig, Daniel R.; Hore, P. J.; Mackenzie, Stuart R., E-mail: christiane.timmel@chem.ox.ac.uk, E-mail: stuart.mackenzie@chem.ox.ac.uk [Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford (United Kingdom)

2016-08-28

Even though the interaction of a <1 mT magnetic field with an electron spin is less than a millionth of the thermal energy at room temperature (k{sub B}T), it still can have a profound effect on the quantum yields of radical pair reactions. We present a study of the effects of sub-millitesla magnetic fields on the photoreaction of flavin mononucleotide with ascorbic acid. Direct control of the reaction pathway is achieved by varying the rate of electron transfer from ascorbic acid to the photo-excited flavin. At pH 7.0, we verify the theoretical prediction that, apart from a sign change, the form of the magnetic field effect is independent of the initial spin configuration of the radical pair. The data agree well with model calculations based on a Green’s function approach that allows multinuclear spin systems to be treated including the diffusive motion of the radicals, their spin-selective recombination reactions, and the effects of the inter-radical exchange interaction. The protonation states of the radicals are uniquely determined from the form of the magnetic field-dependence. At pH 3.0, the effects of two chemically distinct radical pair complexes combine to produce a pronounced response to ∼500 μT magnetic fields. These findings are relevant to the magnetic responses of cryptochromes (flavin-containing proteins proposed as magnetoreceptors in birds) and may aid the evaluation of effects of weak magnetic fields on other biologically relevant electron transfer processes.

Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information

Directory of Open Access Journals (Sweden)

Hannu Myllykallio

2018-05-01

Full Text Available Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as “uracil methyltransferases,” albeit we prefer to refer to them as (ribo thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin co-factors (FADH2 in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and

CORRELATION OF FLAVIN-CONTAINING MONOOXYGENASE ACTIVITY, ALDICARB TOXICITY AND SALINITY IN THE EURYHALINE FISH, JAPANESE MEDAKA (ORYZIAS LATIPES). (R826109)

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

NARCIS (Netherlands)

Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

2014-01-01

The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

OpenAIRE

Abbas, Charles A.; Sibirny, Andriy A.

2011-01-01

Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pterid...

Reduced Flavin: NMR investigation of N(5-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst

Directory of Open Access Journals (Sweden)

Rüterjans Heinz

2005-11-01

Full Text Available Abstract Background The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases, redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results N(5-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3 and N(5 resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5 signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5 signal and the proton exchange rates are little dependent on the buffer system used. Conclusion The exchange rates allow an estimation of the pKa value of N(5-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5-H protonation (to form N(5+-H2 would be consistent with a role of N(5-H as a base.

Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

Science.gov (United States)

Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

2017-02-01

Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

Science.gov (United States)

Abbas, Charles A.; Sibirny, Andriy A.

2011-01-01

Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

Science.gov (United States)

Abbas, Charles A; Sibirny, Andriy A

2011-06-01

Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B₂] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase.

Science.gov (United States)

Carlin, D A; Bertolani, S J; Siegel, J B

2015-02-11

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase

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Dirk Tischler

2018-04-01

Full Text Available Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1 and a two domain protein (VpStyA2B harboring an epoxidase (A2 and a FAD-reductase (B domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s−1 were determined and resulted in a catalytic efficiency (kcat Km−1 of 0.64 s−1 μM−1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV was mutated. One mutant (AAAAA showed 18.7-fold higher affinity for FAD (kcat Km−1 of 5.21 s−1 μM−1 while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg−1 and 0.46 U mg−1, as well as on benzyl methyl sulfide of 1.62 U mg−1 and 3.11 U mg−1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h and produced dominantly (S-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.

Flavoenzyme-catalyzed oxygenations and oxidations of phenolic compounds

NARCIS (Netherlands)

Moonen, MJH; Fraaije, MW; Rietjens, IMCM; Laane, C; van Berkel, WJH

2002-01-01

Flavin-dependent monooxygenases and oxidases play an important role in the mineralization of phenolic compounds. Because of their exquisite regioselectivity and stereoselectivity, these enzymes are of interest for the biocatalytic production of fine chemicals and food ingredients. In our group, we

Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme

DEFF Research Database (Denmark)

de Rond, Tristan; Stow, Parker; Eigl, Ian

2017-01-01

Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C–H activation and cyclization of prodigi...... of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution....

Forward genetic screen for auxin-deficient mutants by cytokinin

Czech Academy of Sciences Publication Activity Database

Wu, L.; Luo, P.; Di, D.W.; Wang, L.; Wang, M.; Lu, C.K.; Wei, S.D.; Zhang, L.; Zhang, T.Z.; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, G.Q.

2015-01-01

Roč. 5, JUL 6 (2015) ISSN 2045-2322 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : ETHYLENE-INSENSITIVE MUTANTS * YUCCA FLAVIN MONOOXYGENASES * ARABIDOPSIS-THALIANA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.228, year: 2015

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

Energy Technology Data Exchange (ETDEWEB)

Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F. [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom); Lau, Peter C. [National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2 (Canada); Hasegawa, Yoshie; Iwaki, Hiroaki [Kansai University (Japan); Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T. [Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald (Germany); Bourenkov, Gleb [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22607 Hamburg (Germany); Littlechild, Jennifer A., E-mail: j.a.littlechild@exeter.ac.uk [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom)

2015-10-31

The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes. The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

International Nuclear Information System (INIS)

Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F.; Lau, Peter C.; Hasegawa, Yoshie; Iwaki, Hiroaki; Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T.; Bourenkov, Gleb; Littlechild, Jennifer A.

2015-01-01

The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes. The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily

Biosynthetic machinery of ionophore polyether lasalocid: enzymatic construction of polyether skeleton.

Science.gov (United States)

Minami, Atsushi; Oguri, Hiroki; Watanabe, Kenji; Oikawa, Hideaki

2013-08-01

Diversity of natural polycyclic polyethers originated from very simple yet versatile strategy consisting of epoxidation of linear polyene followed by epoxide opening cascade. To understand two-step enzymatic transformations at molecular basis, a flavin containing monooxygenase (EPX) Lsd18 and an epoxide hydrolase (EH) Lsd19 were selected as model enzymes for extensive investigation on substrate specificity, catalytic mechanism, cofactor requirement and crystal structure. This pioneering study on prototypical lasalocid EPX and EH provides insight into detailed mechanism of ionophore polyether assembly machinery and clarified remaining issues for polyether biosynthesis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis of 10-Ethyl Flavin: A Multistep Synthesis Organic Chemistry Laboratory Experiment for Upper-Division Undergraduate Students

Science.gov (United States)

Sichula, Vincent A.

2015-01-01

A multistep synthesis of 10-ethyl flavin was developed as an organic chemistry laboratory experiment for upper-division undergraduate students. Students synthesize 10-ethyl flavin as a bright yellow solid via a five-step sequence. The experiment introduces students to various hands-on experimental organic synthetic techniques, such as column…

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

International Nuclear Information System (INIS)

Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

2009-01-01

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

A putative flavin-containing mono-oxygenase as a marker for certain defense and cell death pathways

DEFF Research Database (Denmark)

Olszak, Brian; Malinovsky, Frederikke Gro; Brodersen, Peter

2006-01-01

to a virulent strain. In addition, a close barley homolog was identified by differential display and shown also be induced by pathogen infection. AtFMO::GUS was induced by the fungal toxin fumonisin B1 and by superoxide generation, but not by treatments with hydrogen peroxide, ozone or nitric oxide. During...

Evaluation of the conserve flavin reductase gene from three ...

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... means of PCR technique. The nucleic acid sequences of the PCR primers were designed using conserved nucleic acid sequences of the flavin reductase enzyme from. Rhodococcus sp. strain IGTS8. The oligonucleotide primers were as follows: 5'-GAA TTC ATG TCT GAC. AAG CCG AAT GCC-3' (forward) ...

Monooxygenase, a novel beta-cypermethrin degrading enzyme from Streptomyces sp.

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Shaohua Chen

Full Text Available The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5-8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe(2+, but strongly inhibited by Ag(+, Al(3+, and Cu(2+. The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection.

Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

International Nuclear Information System (INIS)

O'Connor, Meeghan A.; Koza-Taylor, Petra; Campion, Sarah N.; Aleksunes, Lauren M.; Gu, Xinsheng; Enayetallah, Ahmed E.; Lawton, Michael P.; Manautou, José E.

2014-01-01

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 h later with 600 mg APAP/kg. Livers were obtained 4 or 24 h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430 2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection. - Highlights: • Differential expression of genes in mice resistant to acetaminophen hepatotoxicity. • Increased gene expression of Flavin-containing monooxygenase 3 and Galectin-3. • Decrease in MAT1A expression and compensatory hepatocellular regeneration. • Two distinct gene expression

Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

Energy Technology Data Exchange (ETDEWEB)

O' Connor, Meeghan A., E-mail: meeghan.oconnor@boehringer-ingelheim.com [Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092 (United States); Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, CT 06877-0368 (United States); Koza-Taylor, Petra, E-mail: petra.h.koza-taylor@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Campion, Sarah N., E-mail: sarah.campion@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Aleksunes, Lauren M., E-mail: aleksunes@eohsi.rutgers.edu [Rutgers University, Department of Pharmacology and Toxicology, Environmental and Occupational Health Sciences Institute, Piscataway, NJ 08854 (United States); Gu, Xinsheng, E-mail: xinsheng.gu@uconn.edu [Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092 (United States); Enayetallah, Ahmed E., E-mail: ahmed.enayetallah@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Lawton, Michael P., E-mail: michael.lawton@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Manautou, José E., E-mail: jose.manautou@uconn.edu [Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092 (United States)

2014-01-01

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 h later with 600 mg APAP/kg. Livers were obtained 4 or 24 h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430{sub 2} GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection. - Highlights: • Differential expression of genes in mice resistant to acetaminophen hepatotoxicity. • Increased gene expression of Flavin-containing monooxygenase 3 and Galectin-3. • Decrease in MAT1A expression and compensatory hepatocellular regeneration. • Two distinct gene

Effects of Quinizarin and Five Synthesized Derivatives on Fifth Larval Instar Midgut Ecdysone 20-Monooxygenase Activity of the Tobacco Hornworm Manduca sexta

Directory of Open Access Journals (Sweden)

Christopher A. Drummond

2014-01-01

Full Text Available The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone, and five anthraquinones that were synthesized from quinizarin, namely, 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect model Manduca sexta (tobacco hornworm. This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form. M. sexta fifth larval instar midgut homogenates were incubated with increasing concentrations (10−8 to 10−3 M of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibited I50’s of about 4×10-6 to 6×10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4-anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.

Adaptive and Behavioral Changes in Kynurenine 3-Monooxygenase Knockout Mice: Relevance to Psychotic Disorders.

Science.gov (United States)

Erhardt, Sophie; Pocivavsek, Ana; Repici, Mariaelena; Liu, Xi-Cong; Imbeault, Sophie; Maddison, Daniel C; Thomas, Marian A R; Smalley, Joshua L; Larsson, Markus K; Muchowski, Paul J; Giorgini, Flaviano; Schwarcz, Robert

2017-11-15

Kynurenine 3-monooxygenase converts kynurenine to 3-hydroxykynurenine, and its inhibition shunts the kynurenine pathway-which is implicated as dysfunctional in various psychiatric disorders-toward enhanced synthesis of kynurenic acid, an antagonist of both α7 nicotinic acetylcholine and N-methyl-D-aspartate receptors. Possibly as a result of reduced kynurenine 3-monooxygenase activity, elevated central nervous system levels of kynurenic acid have been found in patients with psychotic disorders, including schizophrenia. In the present study, we investigated adaptive-and possibly regulatory-changes in mice with a targeted deletion of Kmo (Kmo -/- ) and characterized the kynurenine 3-monooxygenase-deficient mice using six behavioral assays relevant for the study of schizophrenia. Genome-wide differential gene expression analyses in the cerebral cortex and cerebellum of these mice identified a network of schizophrenia- and psychosis-related genes, with more pronounced alterations in cerebellar tissue. Kynurenic acid levels were also increased in these brain regions in Kmo -/- mice, with significantly higher levels in the cerebellum than in the cerebrum. Kmo -/- mice exhibited impairments in contextual memory and spent less time than did controls interacting with an unfamiliar mouse in a social interaction paradigm. The mutant animals displayed increased anxiety-like behavior in the elevated plus maze and in a light/dark box. After a D-amphetamine challenge (5 mg/kg, intraperitoneal), Kmo -/- mice showed potentiated horizontal activity in the open field paradigm. Taken together, these results demonstrate that the elimination of Kmo in mice is associated with multiple gene and functional alterations that appear to duplicate aspects of the psychopathology of several neuropsychiatric disorders. Copyright © 2016. Published by Elsevier Inc.

Bound Flavin-Cytochrome Model of Extracellular Electron Transfer in Shewanella oneidensis: Analysis by Free Energy Molecular (Postprint)

Science.gov (United States)

2016-06-06

cathodic conditions, oxidized and reduced heme states were assumed, respectively. The calculated results are summarized in Table 2. The solvation free...reports favor a flavin-bound model, proposing two one- electron reductions of flavin, namely, oxidized (Ox) to semiquinone (Sq) and semiquinone to...hydroquinone (Hq), at anodic and cathodic conditions, respectively. In this work, to provide a mechanistic understanding of riboflavin (RF) binding at

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis.

Science.gov (United States)

Minami, Atsushi; Shimaya, Mayu; Suzuki, Gaku; Migita, Akira; Shinde, Sandip S; Sato, Kyohei; Watanabe, Kenji; Tamura, Tomohiro; Oguri, Hiroki; Oikawa, Hideaki

2012-05-02

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis. © 2012 American Chemical Society

Synthesis of methyl propanoate by Baeyer-Villiger monooxygenases

NARCIS (Netherlands)

van Beek, Hugo L.; Winter, Remko T.; Eastham, Graham R.; Fraaije, Marco W.

2014-01-01

Methyl propanoate is an important precursor for polymethyl methacrylates. The use of a Baeyer-Villiger monooxygenase (BVMO) to produce this compound was investigated. Several BVMOs were identified that produce the chemically non-preferred product methyl propanoate in addition to the normal product

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

NARCIS (Netherlands)

Cankar, K.; van Houwelingen, A.; Bosch, H.J.; Sonke, T.; Bouwmeester, H.; Beekwilder, J.P.

2011-01-01

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene

Discovery of Baeyer-Villiger monooxygenases from photosynthetic eukaryotes

NARCIS (Netherlands)

Beneventi, Elisa; Niero, Mattia; Motterle, Riccardo; Fraaije, Marco; Bergantino, Elisabetta

2013-01-01

Baeyer-Villiger monooxygenases are attractive "green" catalysts able to produce chiral esters or lactones starting from ketones. They can act as natural equivalents of peroxyacids that are the catalysts classically used in the organic synthesis reactions, consisting in the cleavage of C-C bonds with

Development of a physiologically based pharmacokinetic model to predict the effects of flavin-containing monooxygenase 3 (FMO3) polymorphisms on itopride exposure.

Science.gov (United States)

Zhou, Wangda; Humphries, Helen; Neuhoff, Sibylle; Gardner, Iain; Masson, Eric; Al-Huniti, Nidal; Zhou, Diansong

2017-09-01

Itopride, a substrate of FMO3, has been used for the symptomatic treatment of various gastrointestinal disorders. Physiologically based pharmacokinetic (PBPK) modeling was applied to evaluate the impact of FMO3 polymorphism on itopride pharmacokinetics (PK). The Asian populations within the Simcyp simulator were updated to incorporate information on the frequency, activity and abundance of FMO3 enzyme with different phenotypes. A meta-analysis of relative enzyme activities suggested that FMO3 activity in subjects with homozygous Glu158Lys and Glu308Gly mutations (Lys158 and Gly308) in both alleles is ~47% lower than those carrying two wild-type FMO3 alleles. Individuals with homozygous Lys158 and Gly308 mutations account for about 5% of the total population in Asian populations. A CL int of 9 μl/min/pmol was optimised for itopride via a retrograde approach as human liver microsomal results would under-predict its clearance by ~7.9-fold. The developed itopride PBPK model was first verified with three additional clinical studies in Korean and Japanese subjects resulting in a predicted clearance of 52 to 69 l/h, which was comparable to those observed (55 to 88 l/h). The model was then applied to predict plasma concentration-time profiles of itopride in Chinese subjects with wild type or homozygous Lys158 and Gly308 FMO3 genotypes. The ratios of predicted to observed AUC of itopride in subjects with each genotype were 1.23 and 0.94, respectively. In addition, the results also suggested that for FMO3 metabolised drugs with a safety margin of 2 or more, proactive genotyping FMO3 to exclude subjects with homozygous Lys158/Gly308 alleles may not be necessary. Copyright © 2017 John Wiley & Sons, Ltd.

Identification of a Baeyer-Villiger monooxygenase sequence motif

NARCIS (Netherlands)

Fraaije, MW; Kamerbeek, NM; van Berkel, WJH; Janssen, DB; Kamerbeek, Nanne M.; Berkel, Willem J.H. van

2002-01-01

Baeyer-Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C-C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXRXXXW(P/D). Studies with

Induction of liver monooxygenases by annatto and bixin in female rats

Directory of Open Access Journals (Sweden)

A.C.A.X. De-Oliveira

2003-01-01

Full Text Available Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'. Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and ß-Apo-8'-carotenal are known to be potent inducers of CYP1A1, a property not shared by others (ß-carotene, lycopene and lutein. Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin and bixin (95% pure on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight, bixin (250 mg/kg body weight or the vehicle only (corn oil, 3.75 g/kg body weight for 5 consecutive days, or were not treated (untreated control. The activities of aniline-4-hydroxylase (A4H, ethoxycoumarin-O-deethylase (ECOD, ethoxy- (EROD, methoxy- (MROD, pentoxy- (PROD and benzyloxy- (BROD resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg induced EROD (3.8x, MROD (4.2x, BROD (3.3x and PROD (2.4x. Bixin (250 mg/kg was a weaker inducer of EROD (2.7x, MROD (2.3x and BROD (1.9x and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Ligand complex structures of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change.

Science.gov (United States)

Im, Dohyun; Matsui, Daisuke; Arakawa, Takatoshi; Isobe, Kimiyasu; Asano, Yasuhisa; Fushinobu, Shinya

2018-03-01

l-Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 (l-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α-group of l-Lys to produce a keto acid and amide, respectively. l-AAO/MOG exhibits limited specificity for l-amino acid substrates with a basic side chain. We previously determined its ligand-free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l-AAO/MOG complexed with l-Lys, l-ornithine, and l-Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l-Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l-AAO/MOG through this route. The atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1.4.3.2 (l-amino acid oxidase), 1.13.12.2 (lysine 2-monooxygenase).

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph

OpenAIRE

Angela V. Smirnova; Peter F. Dunfield

2018-01-01

Methanotrophs are a specialized group of bacteria that can utilize methane (CH4) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silv...

Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes

Science.gov (United States)

Yeager, Chris M.; Bottomley, Peter J.; Arp, Daniel J.; Hyman, Michael R.

1999-01-01

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min−1 to 2.45 min−1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 μM and 112.3 nmol min−1 mg of protein−1 for ethylene and 32.3 μM and 89.2 nmol min−1 mg of protein−1 for propylene. PMID:9925593

Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase from Vibrio harveyi.

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Valentin Borshchevskiy

Full Text Available Na+-translocating NADH:quinone oxidoreductase (NQR is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium.

Integrating cell-free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase.

Science.gov (United States)

Kwon, Yong-Chan; Oh, In-Seok; Lee, Nahum; Lee, Kyung-Ho; Yoon, Yeo Joon; Lee, Eun Yeol; Kim, Byung-Gee; Kim, Dong-Myung

2013-04-01

Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis. Copyright © 2012 Wiley Periodicals, Inc.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

Science.gov (United States)

Cárcamo, Juan Guillermo; Aguilar, Marcelo N; Carreño, Constanza F; Vera, Tamara; Arias-Darraz, Luis; Figueroa, Jaime E; Romero, Alex P; Alvarez, Marco; Yañez, Alejandro J

2017-01-01

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields. Copyright © 2016 Elsevier Inc. All rights reserved.

Kynurenine pathway metabolic balance influences microglia activity: Targeting kynurenine monooxygenase to dampen neuroinflammation.

Science.gov (United States)

Garrison, Allison M; Parrott, Jennifer M; Tuñon, Arnulfo; Delgado, Jennifer; Redus, Laney; O'Connor, Jason C

2018-08-01

Chronic stress or inflammation increases tryptophan metabolism along the kynurenine pathway (KP), and the generation of neuroactive kynurenine metabolites contributes to subsequent depressive-like behaviors. Microglia regulate KP balance by preferentially producing oxidative metabolites, including quinolinic acid. Research has focused on the interplay between cytokines and HPA axis-derived corticosteroids in regulating microglial activity and effects of KP metabolites directly on neurons; however, the potential role that KP metabolites have directly on microglial activity is unknown. Here, murine microglia were stimulated with lipopolysaccharide(LPS). After 6 h, mRNA expression of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α and inducible nitric oxide synthase(iNOS) was dose-dependently increased along with the rate-limiting enzymes for oxidative KP metabolism, indoleamine-2,3-dioxygenase(IDO)-1 and kynurenine 3-monooxygenase(KMO). By 24 h post-LPS, kynurenine and quinolinic acid in the media was elevated. Inhibiting KMO with Ro 61-8048 during LPS challenge attenuated extracellular nitrite accumulation and expression of KMO and TNF-α in response to LPS. Similarly, primary microglia isolated from KMO -/- mice exhibited a significantly reduced pro-inflammatory response to LPS compared to WT controls. To determine whether the substrate (kynurenine) or end product (quinolinic acid) of KMO-dependent metabolism modulates the LPS response, microglia were treated with increasing concentrations of L-kynurenine or quinolinic acid in combination with LPS or saline. Interestingly, quinolinic acid did not impact the microglial LPS response. However, L-kynurenine had dose-dependent inhibitory effect on the LPS response. These data are the first to show an anti-inflammatory effect of KMO inhibition on microglia during immune challenge and suggest that KP metabolic balance may play a direct role in regulating microglia activity. Published by Elsevier Ltd.

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

Science.gov (United States)

Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

2015-05-01

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

NARCIS (Netherlands)

Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The

Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

International Nuclear Information System (INIS)

Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

1985-01-01

The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H 2 O 2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization

Nanoporous Mo2C functionalized 3D carbon architecture anode for boosting flavins mediated interfacial bioelectrocatalysis in microbial fuel cells

Science.gov (United States)

Zou, Long; Lu, Zhisong; Huang, Yunhong; Long, Zhong-er; Qiao, Yan

2017-08-01

An efficient microbial electrocatalysis in microbial fuel cells (MFCs) needs both high loading of microbes (biocatalysts) and robust interfacial electron transfer from microbes to electrode. Herein a nanoporous molybdenum carbide (Mo2C) functionalized carbon felt electrode with rich 3D hierarchical porous architecture is applied as MFC anode to achieve superior electrocatalytic performance. The nanoporous Mo2C functionalized anode exhibits strikingly improved microbial electrocatalysis in MFCs with 5-fold higher power density and long-term stability of electricity production. The great enhancement is attributed to the introduction of rough Mo2C nanostructural interface into macroporous carbon architecture for promoting microbial growth with great excretion of endogenous electron shuttles (flavins) and rich available nanopores for enlarging electrochemically active surface area. Importantly, the nanoporous Mo2C functionalized anode is revealed for the first time to have unique electrocatalytic activity towards redox reaction of flavins with more negative redox potential, indicating a more favourable thermodynamic driving force for anodic electron transfer. This work not only provides a promising electrode for high performance MFCs but also brings up a new insight into the effect of nanostructured materials on interfacial bioelectrocatalysis.

Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

Science.gov (United States)

Carvalho, Alexandra T P; Dourado, Daniel F A R; Skvortsov, Timofey; de Abreu, Miguel; Ferguson, Lyndsey J; Quinn, Derek J; Moody, Thomas S; Huang, Meilan

2017-10-11

Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH

DEFF Research Database (Denmark)

Frandsen, Kristian Erik Høpfner; Poulsen, Jens-Christian Navarro; Tandrup, Tobias

2017-01-01

Lytic polysaccharide monooxygenases (LPMOs) have been found to be key components in microbial (bacterial and fungal) degradation of biomass. They are copper metalloenzymes that degrade polysaccharides oxidatively and act in synergy with glycoside hydrolases. Recently crystallographic studies...

Activation of р-450-depended monooxygenases changing immunotoxicity of phosphoroorganic compounds due to their metabolism character

Directory of Open Access Journals (Sweden)

P.F. Zabrodsky

2010-03-01

Full Text Available It was established that the application of the monooxygenase system inductors (MSI of phenobarbital and benzonal up to acute poisoning of animals by trichlorfom in a dose of 1,0 LD50, metabolized in the organism till production of compounds with higher toxicity caused its immunotoxic properties increase. The experiment was carried out on outbred white rats. the acute dimethyldichlorvinylphosphate (1,0 LD50 poisoning, biotransformation of which proceeded with formation of less-toxic and non-toxic compounds after MSI introduction, caused its decrease of suppression influence on immunity system indices

Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase

DEFF Research Database (Denmark)

Pierce, Brian; Wittrup Agger, Jane; Wichmann, Jesper

2017-01-01

The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of ...

Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology.

Science.gov (United States)

Parrott, Jennifer M; O'Connor, Jason C

2015-01-01

Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO), is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are upregulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s) by which alterations in KMO activity are able to impair neuronal function, and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative, and neuropsychiatric diseases.

Kynurenine 3-monooxygenase: an influential mediator of neuropathology

Directory of Open Access Journals (Sweden)

Jennifer M Parrott

2015-08-01

Full Text Available Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO, is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are up-regulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s by which alterations in KMO activity are able to impair neuronal function and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative and neuropsychiatric diseases.

Active site architecture of a sugar N-oxygenase.

Science.gov (United States)

Thoden, James B; Branch, Megan C; Zimmer, Alex L; Bruender, Nathan A; Holden, Hazel M

2013-05-14

KijD3 is a flavin-dependent N-oxygenase implicated in the formation of the nitro-containing sugar d-kijanose, found attached to the antibiotic kijanimicin. For this investigation, the structure of KijD3 in complex with FMN and its dTDP-sugar substrate was solved to 2.1 Å resolution. In contrast to the apoenzyme structure, the C-terminus of the protein becomes ordered and projects into the active site cleft [Bruender, N. A., Thoden, J. B., and Holden, H. M. (2010) Biochemistry 49, 3517-3524]. The amino group of the dTDP-aminosugar that is oxidized is located 4.9 Å from C4a of the flavin ring. The model provides a molecular basis for understanding the manner in which KijD3 catalyzes its unusual chemical transformation.

Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b.

Science.gov (United States)

Farhan Ul-Haque, Muhammad; Kalidass, Bhagyalakshmi; Vorobev, Alexey; Baral, Bipin S; DiSpirito, Alan A; Semrau, Jeremy D

2015-04-01

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells

DEFF Research Database (Denmark)

Ayesh Hafez Ali, Dalia; Abuelreich, Sarah; Alkeraishan, Nora

2018-01-01

during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor......Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling...... signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand...

Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

Science.gov (United States)

Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

2005-01-01

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

Desaturation reactions catalyzed by soluble methane monooxygenase.

Science.gov (United States)

Jin, Y; Lipscomb, J D

2001-09-01

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.

Marinobacterium sp. strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins.

Science.gov (United States)

Hirano, Hiroyuki; Yoshida, Takako; Fuse, Hiroyuki; Endo, Takayuki; Habe, Hiroshi; Nojiri, Hideaki; Omori, Toshio

2003-06-01

Marinobacterium sp. strain DMS-S1 is a unique marine bacterium that can use dimethyl sulphide (DMS) as a sulphur source only in the presence of light. High-performance liquid chromatography (HPLC) analyses of the culture supernatant revealed that excreted factors, which could transform DMS to dimethyl sulphoxide (DMSO) under light, are FAD and riboflavin. In addition, FAD appeared to catalyse the photolysis of DMS to not only DMSO but also methanesulphonate (MSA), formate, formaldehyde and sulphate. As strain DMS-S1 can use sulphate and MSA as a sole sulphur source independently of light, the excretion of flavins appeared to support the growth on DMS under light. Furthermore, three out of 12 marine bacteria from IAM culture collection were found to be able to grow on DMS with the aid of photolysis by the flavins excreted. This is the first report that bacteria can use light to assimilate oceanic organic sulphur compounds outside the cells by excreting flavins as photosensitizers.

The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

NARCIS (Netherlands)

Heuts, Dominic P. H. M.; Winter, Remko T.; Damsma, Gerke E.; Janssen, Dick B.; Fraaije, Marco W.

2008-01-01

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His(94) and Cys(154). The functional role of this unusual bi-covalent flavin-protein linkage was

Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

International Nuclear Information System (INIS)

Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

2004-01-01

Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

Science.gov (United States)

Morrison, Eliot; Kantz, Auric; Gassner, George T.; Sazinsky, Matthew H.

2013-01-01

The two–component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH– and FAD–dependent epoxidation of styrene to styrene oxide. In this study we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to epoxidase (NSMOA) component and report our findings in light of the 2.2–Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH→FADox charge–transfer intermediate and catalyzes a hydride–transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s−1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium–binding studies indicate that NSMOA binds FADhq ~13–times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)–hydroperoxide intermediate. The half–life of apoSMOB generated in the FAD–transfer reaction is increased ~21–fold, supporting the model of a protein–protein interaction between apoSMOB and NSMOA with the peroxide intermediate. The mechanisms of FAD–dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. Based on these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S–state) and more reactive, transfer state (T–state). Dissociation of NAD+ after the hydride transfer–reaction transiently populates the T–state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB–FADhq shifts the FADhq–binding equilibrium from the T–state to the S–state. Additionally, the 2.2–Å crystal structure of SMOB–FADox reported in this work is discussed in light of the pyridine nucleotide–gated flavin–transfer and electron�

Evidence from Studies with Acifluorfen for Participation of a Flavin-Cytochrome Complex in Blue Light Photoreception for Phototropism of Oat Coleoptiles 12

Science.gov (United States)

Leong, Ta-Yan; Briggs, Winslow R.

1982-01-01

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor. PMID:16662593

Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro

KAUST Repository

Mulaudzi, Takalani

2011-09-01

While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O 2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b.

Science.gov (United States)

Zhang, Tingting; Zhou, Jiti; Wang, Xiaowei; Zhang, Yu

2017-02-01

The coupled effects of nitrogen source and methane monooxygenase (MMO) on the growth and poly-β-hydroxybutyrate (PHB) accumulation capacity of methanotrophs were explored. The ammonia-supplied methanotrophs expressing soluble MMO (sMMO) grew at the highest rate, while N 2 -fixing bacteria expressing particulate MMO (pMMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing pMMO, which might cause their slightly lower growth rate. The highest PHB content (51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing pMMO. Ammonia-supplied bacteria also accumulated a higher content of PHB (45.2%) with the expression of pMMO, while N 2 -fixing bacteria containing pMMO only showed low PHB production capacity (32.1%). The maximal PHB contents of bacteria expressing sMMO were low, with no significant change under different nitrogen source conditions. The low MMO activity, low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N 2 with the expression of pMMO were greatly improved in the cyclic NO 3 - N 2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO. Copyright © 2016. Published by Elsevier B.V.

Structures of the Apo and FAD-bound forms of 2-hydroxybiphenyl 3-monooxygenase (HbpA) locate activity hotspots identified by using directed evolution.

Science.gov (United States)

Jensen, Chantel N; Mielke, Tamara; Farrugia, Joseph E; Frank, Annika; Man, Henry; Hart, Sam; Turkenburg, Johan P; Grogan, Gideon

2015-04-13

The FAD-dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2-hydroxybiphenyl (2HBP) to 2,3-dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed-evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD-complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate-binding site. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of free and bound riboflavin in cow's milk using a novel flavin-binding protein.

Science.gov (United States)

Koop, Julia; Monschein, Stefanie; Pauline Macheroux, E; Knaus, Tanja; Macheroux, Peter

2014-03-01

A recently described putative protease from the gut bacterium Bacteroides thetaiotaomicron (termed ppBat) exhibits two tryptophan residues in the interface which enable specific binding of the isoalloxazine heterocycle of riboflavin and its two cofactor forms, FMN and FAD. Recombinant ppBat was used to capture riboflavin from bovine milk directly without any prior preparation steps. The flavin-loaded protein was then re-isolated by means of affinity chromatography to identify and quantify the captured flavins. Free riboflavin concentrations were determined to 197 and 151μg/l for milk with 3.5% and 0.5% fat content, respectively. Total riboflavin concentrations were also determined after acid-treatment of milk and were 4-5 times higher than for free riboflavin. Free FMN and FAD were not detectable and only trace amounts of FMN were found in milk following acid treatment. The method appears to be amenable to develop a direct assay for free riboflavin in milk and other foods. Copyright © 2013 Elsevier Ltd. All rights reserved.

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

OpenAIRE

Cankar, K.; Houwelingen, van, A.M.M.L.; Bosch, H.J.; Sonke, Th.; Bouwmeester, H.J.; Beekwilder, M.J.

2011-01-01

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-tr...

Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration

Science.gov (United States)

Zwilling, Daniel; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Guidetti, Paolo; Wu, Hui-Qiu; Lee, Jason; Truong, Jennifer; Andrews-Zwilling, Yaisa; Hsieh, Eric W.; Louie, Jamie Y.; Wu, Tiffany; Scearce-Levie, Kimberly; Patrick, Christina; Adame, Anthony; Giorgini, Flaviano; Moussaoui, Saliha; Laue, Grit; Rassoulpour, Arash; Flik, Gunnar; Huang, Yadong; Muchowski, Joseph M.; Masliah, Eliezer; Schwarcz, Robert; Muchowski, Paul J.

2011-01-01

SUMMARY Metabolites in the kynurenine pathway of tryptophan degradation are thought to play an important role in neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease. Metabolites that cause glutamate receptor-mediated excitotoxicity and free radical formation are elevated in the blood and vulnerable brain regions in these diseases, while levels of the neuroprotective metabolite kynurenic acid are often decreased. Here we describe the synthesis and characterization of JM6, a novel small-molecule pro-drug inhibitor of kynurenine 3-monooxygenase (KMO). JM6 raises kynurenic acid and reduces extracellular glutamate in the brain after chronic oral administration by inhibiting KMO in blood. In a transgenic mouse model of Alzheimer’s disease, JM6 prevented spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extended life span, prevented synaptic loss, and decreased microglial activation in a mouse model of Huntington’s disease. These findings support a critical link between blood cells and neurodegeneration that is mediated by KMO and the kynurenine pathway. PMID:21640374

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Directory of Open Access Journals (Sweden)

Christopher F Bennett

2017-03-01

Full Text Available Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt and extends lifespan. Here we report that transaldolase (tald-1 deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2. We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

Directory of Open Access Journals (Sweden)

Faccio Greta

2010-08-01

Full Text Available Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Conclusions Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

Science.gov (United States)

Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

2010-08-20

Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Downregulated kynurenine 3-monooxygenase gene expression and enzyme activity in schizophrenia and genetic association with schizophrenia endophenotypes.

Science.gov (United States)

Wonodi, Ikwunga; Stine, O Colin; Sathyasaikumar, Korrapati V; Roberts, Rosalinda C; Mitchell, Braxton D; Hong, L Elliot; Kajii, Yasushi; Thaker, Gunvant K; Schwarcz, Robert

2011-07-01

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, is an antagonist at N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors and modulates glutamate, dopamine, and acetylcholine signaling. Cortical kynurenic acid concentrations are elevated in the brain and cerebrospinal fluid of schizophrenia patients. The proximal cause may be an impairment of kynurenine 3-monooxygenase (KMO), a rate-limiting enzyme at the branching point of the kynurenine pathway. To examine KMO messenger RNA expression and KMO enzyme activity in postmortem tissue from the frontal eye field (FEF; Brodmann area 6) obtained from schizophrenia individuals compared with healthy control individuals and to explore the relationship between KMO single-nucleotide polymorphisms and schizophrenia oculomotor endophenotypes. Case-control postmortem and clinical study. Maryland Brain Collection, outpatient clinics. Postmortem specimens from schizophrenia patients (n = 32) and control donors (n = 32) and a clinical sample of schizophrenia patients (n = 248) and healthy controls (n = 228). Comparison of quantitative KMO messenger RNA expression and KMO enzyme activity in postmortem FEF tissue between schizophrenia patients and controls and association of KMO single-nucleotide polymorphisms with messenger RNA expression in postmortem FEF and schizophrenia and oculomotor endophenotypes (ie, smooth pursuit eye movements and oculomotor delayed response). In postmortem tissue, we found a significant and correlated reduction in KMO gene expression and KMO enzyme activity in the FEF in schizophrenia patients. In the clinical sample, KMO rs2275163 was not associated with a diagnosis of schizophrenia but showed modest effects on predictive pursuit and visuospatial working memory endophenotypes. Our results provide converging lines of evidence implicating reduced KMO activity in the etiopathophysiology of schizophrenia and related neurocognitive deficits.

A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1.

Science.gov (United States)

Neumann, Christopher S; Walsh, Christopher T; Kay, Robert R

2010-03-30

Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate gene for this transformation. In this work, we present in vivo and in vitro evidence that chlA from Dd encodes a flavin-dependent halogenase capable of catalyzing both chlorinations in the biosynthesis of DIF-1. The results provide in vitro characterization of a eukaryotic oxygen-dependent halogenase and demonstrate a broad reach in biology for this molecular tailoring strategy, notably its involvement in the differentiation program of a social amoeba.

CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes

Science.gov (United States)

Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was fo...

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

Science.gov (United States)

Rafii, F; Franklin, W; Cerniglia, C E

1990-07-01

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.

Molecular simulations in electrochemistry : Electron and proton transfer reactions mediated by flavins in different molecular environments

NARCIS (Netherlands)

Kılıç, M.

2014-01-01

The aim of this thesis is to address specific questions about the role of solvent reorganization on electron transfer in different environments and about the calculation of acidity constant, as well. Particularly, we focus on molecular simulation of flavin in water and different protein (BLUF and

Short range photoinduced electron transfer in proteins: QM-MM simulations of tryptophan and flavin fluorescence quenching in proteins

International Nuclear Information System (INIS)

Callis, Patrik R.; Liu Tiqing

2006-01-01

Hybrid quantum mechanical-molecular mechanics (dynamics) were performed on flavin reductase (Fre) and flavodoxin reductase (Fdr), both from Escherichia coli. Each was complexed with riboflavin (Rbf) or flavin mononucleotide (FMN). During 50 ps trajectories, the relative energies of the fluorescing state (S 1 ) of the isoalloxazine ring and the lowest charge transfer state (CT) were assessed to aid prediction of fluorescence lifetimes that are shortened due to quenching by electron transfer from tyrosine. The simulations for the four cases display a wide range in CT-S 1 energy gap caused by the presence of phosphate, other charged and polar residues, water, and by intermolecular separation between donor and acceptor. This suggests that the Gibbs energy change (ΔG 0 ) and reorganization energy (λ) for the electron transfer may differ in different flavoproteins

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

DEFF Research Database (Denmark)

Li, Jing; Kristiansen, Kim A.; Hansen, Bjarne Gram

2011-01-01

the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular...

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

Science.gov (United States)

Smirnova, Angela V; Dunfield, Peter F

2018-03-06

Methanotrophs are a specialized group of bacteria that can utilize methane (CH₄) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1) The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris ; (2) Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata ; (3) The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris . These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata .

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

Science.gov (United States)

Rafii, F; Franklin, W; Cerniglia, C E

1990-01-01

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly. Images PMID:2202258

Expression, purification and characterization of human Dopamine ß-monooxygenase

DEFF Research Database (Denmark)

Vendelboe, Trine Vammen

catalytic domains called ascorbate dependent type IImonooxygenase domains and a C-terminal dimerization domain. DBM is related to peptidylglycine a-hydroxylating monooxygenase (PHM). They are 28 % identical over approximately 300 amino scids (AA) which corresponds to the catalytic domains. This is, among...... residue 47-596 in each chain, was hereafter manually built. The structure reveals the first structural insights into the DOMON domain and the C-terminal dimerization domain and it shows two different conformations of the catalytic domains. An open conformation, that resembles the structures known from PHM...

Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

Directory of Open Access Journals (Sweden)

Mohd Firdaus Abdul-Wahab

2012-12-01

Full Text Available Citrobacter freundii A1 isolated from a sewage treatment facility was demonstrated to be able to effectively decolorize azo dyes as pure and mixed culture. This study reports on the investigation on the enzymatic systems involved. An assay performed suggested the possible involvement of flavin reductase (Fre as an azo reductase. A heterologouslyexpressed recombinant Fre from C. freundii A1 was used to investigate its involvement in the azo reduction process. Three model dyes were used, namely Acid Red 27 (AR27, Direct Blue 15 (DB15 and Reactive Black 5 (RB5. AR27 was found to be reduced the fastest by Fre, followed by RB5, and lastly DB15. Redox mediators nicotinamide adenine dinucleotide (NADH and riboflavin enhance the reduction, suggesting the redox activity of the enzyme. The rate and extent of reduction of the model dyes correlate well with the reduction potentials (Ep. The data presented here strongly suggest that Fre is one of the enzymes responsible for azo reduction in C. freundii A1, acting via an oxidation-reduction reaction.

LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

OpenAIRE

Manns, M P; Griffin, K J; Sullivan, K F; Johnson, E F

1991-01-01

LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA...

Redox Reactions of Reduced Flavin Mononucleotide (FMN), Riboflavin (RBF), and Anthraquinone-2,6-disulfonate (AQDS) with Ferrihydrite and Lepidocrocite

Energy Technology Data Exchange (ETDEWEB)

Shi, Zhi [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Zachara, John M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Shi, Liang [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Wang, Zheming [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Moore, Dean A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kennedy, David W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Fredrickson, Jim K. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2012-09-17

Flavins are secreted by the dissimilatory iron-reducing bacterium Shewanella and can function as endogenous electron transfer mediators (ETM). In order to assess the potential importance of flavins in Fe(III) bioreduction, we investigated the redox reaction kinetics of reduced flavins (FMNH2 and RBFH2) with ferrihydrite and lepidocrocite. The organic reductants rapidly reduced and dissolved ferrihydrite and lepidocrocite in the pH range 4-8. The rate constant k for 2-line ferrihydrite reductive dissolution by FMNH2 was 87.5 ± 3.5 M-1∙s-1 at pH 7.0 in batch reactors, and the k was similar for RBFH2. For lepidocrocite, the k was 500 ± 61 M-1∙s-1 for FMNH2, and 236 ± 22 M-1∙s-1 for RBFH2. The surface area normalized initial reaction rates (ra) were between 0.08 and 77 μmoles∙m-2∙s-1 for various conditions in stopped-flow experiments. Initial rates (ro) were first-order with respect to Fe(III) oxide concentration, and ra increased with decreasing pH. Poorly crystalline 2-line ferrihydrite yielded the highest ra, followed by more crystalline 6-line ferrihydrite, and crystalline lepidocrocite. Compared to a previous whole-cell study with Shewanella oneidensis strain MR-1, our findings suggest that ETM reduction by the Mtr pathway coupled to lactate oxidation are rate limiting, rather than heterogeneous electron transfer to the Fe(III) oxide.

Fundamental role of Methylenetetrahydrofolate Reductase 677 C->T genotype and Flavin compounds in biochemical phenotypes for schizophrenia and schizoaffective psychosis.

Directory of Open Access Journals (Sweden)

Stephanie Fryar-Williams

2016-11-01

Full Text Available The Mental Health Biomarker Project (2010-2016 explored variables for psychosis in schizophrenia and schizoaffective disorder. Blood samples from 67, highly-characterized symptomatic cases and 67 gender and age matched control participants were analysed for methyl tetrahydrofolate reductase (MTHFR 677C->T gene variants and for vitamin B6, B12 and D, folate, unbound copper, zinc cofactors for enzymes in the methylation cycle and related catecholamine pathways. Urine samples were analysed for indole-catecholamines, their metabolites and oxidative-stress marker, hydroxylpyrolline-2-one (HPL. Rating scales were Brief Psychiatric Rating Scale, Positive and Negative Syndrome Scale, Global Assessment of Function scale, Clinical Global Impression score and Social and Occupational Functioning Scale. Analysis used Spearman’s correlates, Receiver Operating Characteristics and structural equation modelling (SEM. The correlative pattern of variables in the overall participant sample strongly implicated Monoamine Oxidase (MAO enzyme inactivity so the significant role of MAO’s cofactor flavin adenine nucleotide (FAD and its precursor flavin adenine mononucleotide (FMN within the biochemical pathways was investigated and confirmed as 70% on SEM of the total sample. Splitting the data sets for MTHFR 677C->T polymorphism variants coding for the MTHFR enzyme, discovered that biochemistry variables relating to the wild-type enzyme differed markedly in pattern from those coded by the homozygous variant and that the hereozygous-variant pattern resembled the wild type-coded pattern. The MTHFR 677C->T -wild and -heterozygous gene variants have a pattern of depleted vitamin cofactors characteristic of flavin insufficiency with under-methylation and severe oxidative stress. The second homozygous MTHFR 677TT pattern related to elevated copper:zinc ratio and a vitamin pattern related to flavin sufficiency and risk of over-methylation. The two gene variants and their

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph

Directory of Open Access Journals (Sweden)

Angela V. Smirnova

2018-03-01

Full Text Available Methanotrophs are a specialized group of bacteria that can utilize methane (CH4 as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO, of either a soluble, cytoplasmic type (sMMO, or a particulate, membrane-bound type (pMMO. Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1 The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris; (2 Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata; (3 The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris. These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata.

Effect of PCB 126 on aryl hydrocarbon receptor 1 (AHR1) and AHR1 nuclear translocator 1 (ARNT1) mRNA expression and CYP1 monooxygenase activity in chicken (Gallus domesticus) ovarian follicles.

Science.gov (United States)

Wójcik, Dagmara; Antos, Piotr A; Katarzyńska, Dorota; Hrabia, Anna; Sechman, Andrzej

2015-12-03

The aim of the experiment was to study the in vitro effect of 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) on aryl hydrocarbon receptor (AHR1) and AHR1 nuclear translocator (ARNT1) mRNA expression and the activity of CYP1 family monooxygenases in chicken ovarian follicles. White (1-4 mm) and yellowish (4-8 mm) prehierarchical follicles as well as fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were incubated in a medium supplemented with 0 (control group), 1, 10 or 100 nM PCB 126. The incubation was carried out for 6 h or 24 h for determination of mRNA expression of AHR1 and ARNT1 genes (real-time qPCR) and CYP1 monooxygenase activity (EROD and MROD fluorometric assays), respectively. It was found that chicken ovarian follicles express mRNA of AHR1 and ARNT1 genes. A modulatory effect of PCB 126 on AHR1 and ARNT1 expression depended not only on the biphenyl concentration but also on the follicular layer and the maturational state of the follicle. EROD and MROD activities appeared predominantly in the granulosa layer of the yellow preovulatory follicles. PCB 126 induced these activities in a dose-dependent manner in all ovarian follicles. The obtained results suggest that ovarian follicles, especially the granulosa layer, are involved in the detoxification process of PCBs in the laying hen. Taking this finding into consideration it can be suggested that the granulosa layer of the yellow hierarchical follicles plays a key role in the protective mechanism which reduces the amount of transferred dioxin-like compounds into the yolk of the oocyte. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mechanism of flavin reduction in the class 1A dihydroorotate dehydrogenase from Lactococcus lactis

DEFF Research Database (Denmark)

Fagan, Rebecca L; Jensen, Kaj Frank; Björnberg, Olof

2007-01-01

is concerted or stepwise was addressed for the class 1A enzyme from Lactococcus lactis by determining kinetic isotope effects (KIEs) on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined at two pH values. At pH 7.0, KIEs were approximately 2-fold for DHO labeled singly...... at the 5-position or the 6-position and approximately 4-fold for DHO labeled at both the 5- and 6-positions. At pH 8.5, the KIEs observed for DHO labeled at the 5-position, the 6-position, and the 5- and 6-positions were approximately 2-, approximately 3-, and approximately 6-fold, respectively....... These isotope effects are consistent with a concerted oxidation of DHO. The pH dependence of reduction was also determined, and a pKa of 8.3 was found. This pKa can be attributed to the ionization of the active site cysteine which deprotonates C5 of DHO during the reaction. To further investigate the importance...

Exploring flavin-containing carbohydrate oxidases

NARCIS (Netherlands)

Ferrari, Alessandro Renato

2017-01-01

Oxidases are enzymes capable of removing one or more electrons from their substrate and transfer them to molecular oxygen, forming hydrogen peroxide. Due to their high regio- and enantioselectivity, their use is preferred over traditional organic chemistry methods. Among the oxidases, flavoprotein

[Association of kynurenine-3-monooxygenase gene with schizophrenia].

Science.gov (United States)

Golimbet, V E; Lezheiko, T V; Alfimova, M V; Abramova, L I; Kondrat'ev, N V

2014-06-01

Neurotoxic products produced during tryptophan metabolism via the kynurenine pathway could be involved in schizophrenia pathogenesis. It has been shown that kynurenine-3-monooxygenase (KMO) is indirectly involved in these products' formation. KMO polymorphic loci rs2275163 (C/T) and rs1053230 (A/G) were examined in 187 schizophrenia patients and 229 healthy subjects. A genetic combination of allele T and genotype GG was observed more often in a patient group compared with healthy controls (p = 0.003, OR 2.0 (95% CI 1.2-2.9). In the latter group, this combination was associated with schizophrenia endophenotype (p = 0.04), which manifested in a higher expression of schizotypal personality traits assessed using the MMPI test.

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis.

Science.gov (United States)

Luthra, Pratibha Mehta; Singh, Satendra

2010-05-01

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which L: -dopa is formed from the substrate L-tyrosine. L-Dopa concentration and activity of L-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum L-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH(4), and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K (m) value of 808.63 microM for L-tyrosine at 37 degrees C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 microM L-tyrosine. Higher concentrations of the cofactor 6-MPH(4), however, completely inhibited the synthesis of L-dopa. Tyrosine hydroxylase converted specific monophenols such as L-tyrosine (808.63 microM) and tyramine (K (m) 1.1 mM) to diphenols L-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.

Coupled reactions by coupled enzymes : alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions

NARCIS (Netherlands)

Aalbers, Friso S; Fraaije, Marco W

2017-01-01

The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP(+) to oxidize cyclohexanol to form cyclohexanone and NADPH. Both

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

Science.gov (United States)

Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Thomas, Marian A. R.; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J.

2013-01-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD+, did not differ between Kmo−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease. PMID:24189070

HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch.

Science.gov (United States)

Kline, Chelsey D; Mayfield, Mary; Blackburn, Ninian J

2013-04-16

Peptidylglycine monooxygenase is a copper-containing enzyme that catalyzes the amidation of neuropeptides hormones, the first step of which is the conversion of a glycine-extended pro-peptide to its α-hydroxyglcine intermediate. The enzyme contains two mononuclear Cu centers termed CuM (ligated to imidazole nitrogens of H242, H244 and the thioether S of M314) and CuH (ligated to imidazole nitrogens of H107, H108, and H172) with a Cu-Cu separation of 11 Å. During catalysis, the M site binds oxygen and substrate, and the H site donates the second electron required for hydroxylation. The WT enzyme shows maximum catalytic activity at pH 5.8 and undergoes loss of activity at lower pHs due to a protonation event with a pKA of 4.6. Low pH also causes a unique structural transition in which a new S ligand coordinates to copper with an identical pKA, manifest by a large increase in Cu-S intensity in the X- ray absorption spectroscopy. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819-10828), we tentatively assigned the new Cu-S interaction to binding of M109 to the H-site (part of an HHM conserved motif common to all but one member of the family). Here we follow up on these findings via studies on the catalytic activity, pH-activity profiles, and spectroscopic (electron paramagnetic resonance, XAS, and Fourier transform infrared) properties of a number of H-site variants, including H107A, H108A, H172A, and M109I. Our results establish that M109 is indeed the coordinating ligand and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent. The histidine mutants show more complex behavior, but the almost complete lack of activity in all three variants coupled with only minor differences in their spectroscopic properties suggests that unique structural elements at H are critical for functionality. The data suggest a more general

Cell-secreted flavins bound to membrane cytochromes dictate electron transfer reactions to surfaces with diverse charge and pH.

Science.gov (United States)

Okamoto, Akihiro; Kalathil, Shafeer; Deng, Xiao; Hashimoto, Kazuhito; Nakamura, Ryuhei; Nealson, Kenneth H

2014-07-11

The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.

Kynurenine 3-monooxygenase polymorphisms: relevance for kynurenic acid synthesis in patients with schizophrenia and healthy controls

DEFF Research Database (Denmark)

Holtze, Maria; Saetre, Peter; Engberg, Göran

2012-01-01

on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. Methods: We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were...... selected covering KMO and were analyzed in UNPHASED. Results: We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA....... Limitations: Given the limited sample size, the results are tentative until replication. Conclusion: Our results suggest that the nonsynonymous KMO SNP rs1053230 influences CSF concentrations of KYNA....

Exploring the Substrate Scope of Baeyer–Villiger Monooxygenases with Branched Lactones as Entry towards Polyesters

NARCIS (Netherlands)

Delgove, Marie; Fürst, Maximilian; Fraaije, Marco; Bernaerts, Katrien; de Wildeman, Stefaan

2018-01-01

Baeyer–Villiger monooxygenases (BVMOs) are biocatalysts that are able to convert cyclic ketones into lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters.

Exploring the substrate scope of Baeyer-Villiger monooxygenases with branched lactones as entry towards polyesters

NARCIS (Netherlands)

Delgove, Marie; Fürst, Maximilian; Fraaije, Marco; Bernaerts, Katrien; De Wildeman, Stefaan M A

2018-01-01

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts able to convert cyclic ketones to lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters.The product

A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

Science.gov (United States)

Chua, Ngee Kiat; Howe, Vicky; Jatana, Nidhi; Thukral, Lipi; Brown, Andrew J

2017-12-08

Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Fundamental Role of Methylenetetrahydrofolate Reductase 677 C → T Genotype and Flavin Compounds in Biochemical Phenotypes for Schizophrenia and Schizoaffective Psychosis

Science.gov (United States)

Fryar-Williams, Stephanie

2016-01-01

The Mental Health Biomarker Project (2010–2016) explored variables for psychosis in schizophrenia and schizoaffective disorder. Blood samples from 67, highly characterized symptomatic cases and 67 gender and age matched control participants were analyzed for methyl tetrahydrofolate reductase (MTHFR) 677C → T gene variants and for vitamin B6, B12 and D, folate, unbound copper, zinc cofactors for enzymes in the methylation cycle, and related catecholamine pathways. Urine samples were analyzed for indole-catecholamines, their metabolites, and oxidative-stress marker, hydroxylpyrolline-2-one (HPL). Rating scales were Brief Psychiatric Rating Scale, Positive and Negative Syndrome Scale, Global Assessment of Function scale, Clinical Global Impression (CGI) score, and Social and Occupational Functioning Assessment Scale (SOFAS). Analysis used Spearman’s correlates, receiver operating characteristics and structural equation modeling (SEM). The correlative pattern of variables in the overall participant sample strongly implicated monoamine oxidase (MAO) enzyme inactivity so the significant role of MAO’s cofactor flavin adenine nucleotide and its precursor flavin adenine mononucleotide (FMN) within the biochemical pathways was investigated and confirmed as 71% on SEM of the total sample. Splitting the data sets for MTHFR 677C → T polymorphism variants coding for the MTHFR enzyme, discovered that biochemistry variables relating to the wild-type enzyme differed markedly in pattern from those coded by the homozygous variant and that the hereozygous-variant pattern resembled the wild-type-coded pattern. The MTHFR 677C → T-wild and -heterozygous gene variants have a pattern of depleted vitamin cofactors characteristic of flavin insufficiency with under-methylation and severe oxidative stress. The second homozygous MTHFR 677TT pattern related to elevated copper:zinc ratio and a vitamin pattern related to flavin sufficiency and risk of over-methylation. The

Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

Energy Technology Data Exchange (ETDEWEB)

Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg (Germany); Tanwar, M.; Veetil, S.K.; Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Stierl, M.; Hegemann, P. [Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany)

2013-09-23

Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

International Nuclear Information System (INIS)

Penzkofer, A.; Tanwar, M.; Veetil, S.K.; Kateriya, S.; Stierl, M.; Hegemann, P.

2013-01-01

Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction

Photo-induced reduction of flavin mononucleotide in aqueous solutions

International Nuclear Information System (INIS)

Song, S.-H.; Dick, B.; Penzkofer, A.

2007-01-01

The photo-induced reduction of flavin mononucleotide (FMN) in aqueous solutions is studied by absorption spectra measurement under aerobic and anaerobic conditions. Samples without exogenous reducing agent and with the exogenous reducing agents ethylene-diamine-tetraacetic acid (EDTA) and dithiothreitol (DTT) are investigated. Under anaerobic conditions the photo-induced reduction with and without reducing agents is irreversible. Under aerobic conditions the photo-reduction without added reducing agent is small compared to the photo-degradation, and the photo-reduction of FMN by the reducing agents is reversible (re-oxidation in the dark). During photo-excitation of FMN the dissolved oxygen is consumed by singlet oxygen formation and subsequent chemical reaction. After light switch-off slow re-oxidation (slow absorption recovery) occurs due to air in-diffusion from surface. EDTA degradation by FMN excitation leads to oxygen scavenging. The quantum efficiencies of photo-reduction under aerobic and anaerobic conditions are determined. The re-oxidation of reduced FMN under aerobic conditions and due to air injection is investigated

Molecular dynamics analysis reveals structural insights into mechanism of nicotine N-demethylation catalyzed by tobacco cytochrome P450 mono-oxygenase.

Directory of Open Access Journals (Sweden)

Shan Wang

Full Text Available CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys-trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.

Effect of transplantation of muscle tissue in rats from the same litter on total number of flavins and FAD

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S. N. Kobylnik

2015-01-01

Full Text Available Riboflavin is a member of redox enzymes involved in fatty acid oxidation and energy generation. Important role of this vitamin is in reproductive function. Exchange of transformation of riboflavin in animal tissues and cells of microorganisms include reactions that lead to synthesis and subsequent collapse of FMN and FAD. It is involved in enhancing antitumor activity of many anticancer drugs, as well as activation of the immune system to kill tumor cells. Issues of transport of riboflavin and its derivatives in animals have been studied enough. Investigations of changes of the balance of riboflavin and its metabolites in muscular tissues before transplantation in rats from one litter and at operation without replanting were conducted, based on the Udenfriend method of flavin determination. Transplantation in the experiment was carried out on white non-linear male rats weighing 180–300 g. Animals were taken out of the experiment by passing electric current through the medulla. Belly muscular tissue was taken from donor rats of the same litter, and that tissue was sewn to homological muscular tissue of the recipient. The same procedure was carried out with femoral muscular tissue. In the course of operation without replanting the same manipulations have been made except for transplantation stage (for determination of the effect of surgical intervention. Tissue not subject to any surgical intervention served as a control. Parameters of the study were measured on the first, third and seventh days after transplantation. Transplantation of muscular tissue caused no changes in total flavin amount. Content of RF + FMN after transplantation of muscular tissue in rats of the same litter decreased in femoral muscular tissue of the recipient. Transplantation of muscular tissues in rats from the same litter lead to increase in FAD amount in femoral muscular tissue of the donor and recipient on the third day of the experiment. Transplantation of femoral

Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

Energy Technology Data Exchange (ETDEWEB)

Wang, Zheming; Shi, Zhi; Shi, Liang; White, Gaye F.; Richardson, David J.; Clarke, Thomas A.; Fredrickson, Jim K.; Zachara, John M.

2015-08-25

Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, some evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin that are proposed to mediate electron transfer (Marsili et al., 2008). In this work, we used methyl viologen (MV•+)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of secreted flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200 µM) by MELs ([MV•+] ~ 42 µM and MtrABC ≤ 1 nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MV•+ and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 seconds. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (≤ 1 µM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. However, at higher FMN concentrations (> 1 µM), the reaction rates for both steps decreased and varied inversely with FMN concentration, indicating that FMN inhibited the MEL to Fe(III)-oxide electron transfer

A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1

OpenAIRE

Neumann, Christopher S.; Walsh, Christopher T.; Kay, Robert R.

2010-01-01

Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate ...

Enantiomeric behaviour of albendazole and fenbendazole sulfoxides in domestic animals: pharmacological implications.

Science.gov (United States)

Capece, Bettencourt P S; Virkel, Guillermo L; Lanusse, Carlos E

2009-09-01

Albendazole and fenbendazole are methylcarbamate benzimidazole anthelmintics extensively used to control gastrointestinal parasites in domestic animals. These parent compounds are metabolised to albendazole sulfoxide and fenbendazole sulfoxide (oxfendazole), respectively. Both sulfoxide derivatives are anthelmintically active and are manufactured for use in animals. They metabolites have an asymmetric centre on their chemical structures and two enantiomeric forms of each sulfoxide have been identified in plasma, tissues of parasite location and within target helminths. Both the flavin-monooxygenase and cytochrome P450 systems are involved in the enantioselective biotransformation of these anthelmintic compounds in ruminant species. A relevant progress on the understanding of the relationship among enantioselective metabolism and systemic availability of each enantiomeric form has been achieved. This article reviews the current knowledge on the pharmacological implications of the enantiomeric behaviour of albendazole sulfoxide and oxfendazole in domestic animals.

Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

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Gao, Jingjing; Yao, Licheng; Xia, Tingting; Liao, Xuebin; Zhu, Deyu; Xiang, Ye

2018-04-01

The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ∼110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-α-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

Fiskelugt--det kunne vaere trimethylaminuri

DEFF Research Database (Denmark)

Haugaard, Line Klingen; Lund, Allan Meldgaard; Patursson, Poula

2010-01-01

We present a case with a ten-year-old girl with trimethylaminuria (TMAU). Primary TMAU is caused by a deficiency of flavin monooxygenase 3 (FMO3) due to mutations in the FMO3-gene. Patients suffering from TMAU show an impaired enzymatic oxidation of fish-smelling trimethylamine, and their excretion...... of this amine in body fluids produces an unpleasant body odour. TMAU is also seen secondary to e.g. liver diseases. It remains unknown if TMAU causes other problems than malodour, and today social and psychological problems are considered the most important consequence. Treatment includes a low-choline diet...

CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase.

Science.gov (United States)

Mitić, Natasa; Schwartz, Jennifer K; Brazeau, Brian J; Lipscomb, John D; Solomon, Edward I

2008-08-12

The multicomponent soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through the activation of O 2 at a nonheme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in the rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with ligand-field (LF) calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O 2 reactivity. MMOB variants that do not greatly increase O 2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O 2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O 2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O 2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO.

Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

OpenAIRE

Bosma, T.; Janssen, D.B.

1998-01-01

Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropr...

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

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Tom A. Ewing

2018-01-01

Full Text Available Vanillyl alcohol oxidase (VAO and eugenol oxidase (EUGO are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol and a EUGO variant (V436I with increased activity towards chavicol (4-allylphenol and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

Science.gov (United States)

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Pam (Peptidylglycine α-amidating monooxygenase) heterozygosity alters brain copper handling with region specificity

Science.gov (United States)

Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

2013-01-01

Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in postsynaptic vesicular fractions. Cu followed a similar pattern, with ~20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) is selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not the hippocampus in PAM+/− mice, along with GABAB receptor mRNA levels. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. PMID:24032518

Photosensitization of human diploid cell cultures by intracellular flavins and protection by antioxidants

International Nuclear Information System (INIS)

Pereira, O.M.; Smith, J.R.; Packer, L.

1976-01-01

The damaging effects of near ultraviolet and visible light on WI-38 human diploid lung fibroblasts were investigated. WI-38 cells in culture were killed by light doses ranging from 2 to 10 x 10 3 W/m 2 h. There was an inverse correlation between culture age, i.e. population doubling level and photosensitivity. However, this effect could not be related to capacity for DNA synthesis and cell division. Flavins were clearly implicated as endogenous photosensitizers, and antioxidants such as d,l-α-tocopherol (vitamin E), BHT and ascorbic acid were found to afford the cells protection from light damage. Furthermore, products of lipid peroxidation could be detected in cell homogenates irradiated in the presence of riboflavin. (author)

Transcriptional control of the isoeugenol monooxygenase of Pseudomonas nitroreducens Jin1 in Escherichia coli.

Science.gov (United States)

Ryu, Ji-Young; Seo, Jiyoung; Ahn, Joong-Hoon; Sadowsky, Michael J; Hur, Hor-Gil

2012-01-01

Vanillin is one of the most valuable compounds in the flavoring and fragrance industries, and many attempts to produce natural vanillin have been made in recent years. Isoeugenol monooxygenase (Iem) converts the phenylpropanoid compound isoeugenol to vanillin. In Pseudomonas nitroreducens Jin1, the positive regulatory protein IemR is divergently expressed from Iem, and the promoter region is located between the genes. In this study, we investigated the transcriptional regulation of iem in Escherichia coli. We focused on inducers and regulatory protein IemR. Transcription of iem was found to be dependent on the amounts of isoeugenol and IemR. Isoeugenol was found to be the best inducer of iem, followed by trans-anethole, which induced iem to 58% of the transcription level observed for isoeugenol. Overproduction of IemR in E. coli significantly increased the transcription of iem, up to 96-fold, even in the absence of isoeugenol, as compared to basally expressed IemR. Results of this study indicate that the transcription of iem iss dependent on the type of inducers and on IemR. They should contribute to the development of bioengineering strategies for increased production of vanillin through high-level expression of the isoeugenol monooxygenase gene in microorganisms.

Development of a series of aryl pyrimidine kynurenine monooxygenase inhibitors as potential therapeutic agents for the treatment of Huntington's disease.

Science.gov (United States)

Toledo-Sherman, Leticia M; Prime, Michael E; Mrzljak, Ladislav; Beconi, Maria G; Beresford, Alan; Brookfield, Frederick A; Brown, Christopher J; Cardaun, Isabell; Courtney, Stephen M; Dijkman, Ulrike; Hamelin-Flegg, Estelle; Johnson, Peter D; Kempf, Valerie; Lyons, Kathy; Matthews, Kimberly; Mitchell, William L; O'Connell, Catherine; Pena, Paula; Powell, Kendall; Rassoulpour, Arash; Reed, Laura; Reindl, Wolfgang; Selvaratnam, Suganathan; Friley, Weslyn Ward; Weddell, Derek A; Went, Naomi E; Wheelan, Patricia; Winkler, Christin; Winkler, Dirk; Wityak, John; Yarnold, Christopher J; Yates, Dawn; Munoz-Sanjuan, Ignacio; Dominguez, Celia

2015-02-12

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase

OpenAIRE

Dedysh, Svetlana N.; Naumoff, Daniil G.; Vorobev, Alexey V.; Kyrpides, Nikos; Woyke, Tanja; Shapiro, Nicole; Crombie, Andrew T.; Murrell, J. Colin; Kalyuzhnaya, Marina G.; Smirnova, Angela V.; Dunfield, Peter F.

2015-01-01

Methyloferula stellata AR4 is an aerobic acidophilic methanotroph, which, in contrast to most known methanotrophs but similar to Methylocella spp., possesses only a soluble methane monooxygenase. However, it differs from Methylocella spp. by its inability to grow on multicarbon substrates. Here, we report the draft genome sequence of this bacterium.

Characterization of Active Site Residues of Nitroalkane Oxidase†

Science.gov (United States)

Valley, Michael P.; Fenny, Nana S.; Ali, Shah R.; Fitzpatrick, Paul F.

2010-01-01

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ~5-fold and decreases in the rate constant for product release of ~2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

Directory of Open Access Journals (Sweden)

Walker M Andrew

2010-07-01

Full Text Available Abstract Background Plant cytochrome P450 monooxygenases (CYP mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines. Results Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS in the upstream region and three candidate polyadenylation (PolyA sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression. Conclusions This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression

Targeting kynurenine 3-monooxygenase (KMO): implications for therapy in Huntington's disease.

Science.gov (United States)

Thevandavakkam, Mathuravani A; Schwarcz, Robert; Muchowski, Paul J; Giorgini, Flaviano

2010-12-01

Huntington's disease (HD) is an adult onset neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. Recent work has shown that perturbation of kynurenine pathway (KP) metabolism is a hallmark of HD pathology, and that changes in brain levels of KP metabolites may play a causative role in this disease. The KP contains three neuroactive metabolites, the neurotoxins 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN), and the neuroprotectant kynurenic acid (KYNA). In model systems in vitro and in vivo, 3-HK and QUIN have been shown to cause neurodegeneration via a combination of excitotoxic mechanisms and oxidative stress. Recent studies with HD patient samples and in HD model systems have supported the idea that a shift away from the synthesis of KYNA and towards the formation of 3-HK and QUIN may trigger the neuropathological features observed in HD. The enzyme kynurenine 3-monooxygenase (KMO) is located at a critical branching point in the KP such that inhibition of this enzyme by either pharmacological or genetic means shifts the flux in the pathway towards the formation of KYNA. This intervention ameliorates disease-relevant phenotypes in HD models. Here we review the work implicating the KP in HD pathology and discuss the potential of KMO as a therapeutic target for this disorder. As several neurodegenerative diseases exhibit alterations in KP metabolism, this concept has broader implications for the treatment of brain diseases.

Sample container for neutron activation analysis

International Nuclear Information System (INIS)

Lersmacher, B.; Verheijke, M.L.; Jaspers, H.J.

1983-01-01

The sample container avoids contaminating the sample substance by diffusion of foreign matter from the wall of the sample container into the sample. It cannot be activated, so that the results of measurements are not falsified by a radioactive container wall. It consists of solid carbon. (orig./HP) [de

Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

Science.gov (United States)

Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

2013-09-01

Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

Science.gov (United States)

Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

2015-04-01

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase.

Science.gov (United States)

Dedysh, Svetlana N; Naumoff, Daniil G; Vorobev, Alexey V; Kyrpides, Nikos; Woyke, Tanja; Shapiro, Nicole; Crombie, Andrew T; Murrell, J Colin; Kalyuzhnaya, Marina G; Smirnova, Angela V; Dunfield, Peter F

2015-03-05

Methyloferula stellata AR4 is an aerobic acidophilic methanotroph, which, in contrast to most known methanotrophs but similar to Methylocella spp., possesses only a soluble methane monooxygenase. However, it differs from Methylocella spp. by its inability to grow on multicarbon substrates. Here, we report the draft genome sequence of this bacterium. Copyright © 2015 Dedysh et al.

Induction of cytochrome P450-associated monooxygenases in northern leopard frogs, Rana pipiens, by 3,3',4,4',5-pentachlorobiphenyl

Science.gov (United States)

Huang, Y.-W.; Melancon, M.J.; Jung, R.E.; Karasov, W.H.

1998-01-01

Northern leopard frogs (Rana pipiens) were injected intraperitoneally either with a solution of polychlorinated biphenyl (PCB) 126 in corn oil at a concentration of 0.2, 0.7, 2.3 and 7.8 mg/kg body weight or with corn oil alone. Appropriate assay conditions with hepatic microsomes were determined for four cytochrome P450-associated monooxygenases: ethoxyresorufin-O-dealkylase (EROD), methoxy-ROD (MROD), benzyloxy-ROD (BROD) and pentoxy-ROD (PROD). One week after PCB administration, the specific activities of EROD, MROD, BROD and PROD were not elevated at doses ? 0.7 mg/kg (p > 0.05), but were significantly increased at doses ? 2.3 mg/kg compared to the control groups (p leopard frogs.

Optimized formation of detergent micelles of beta-carotene and retinal production using recombinant human beta,beta-carotene 15,15'-monooxygenase.

Science.gov (United States)

Kim, Nam-Hee; Kim, Yeong-Su; Kim, Hye-Jung; Oh, Deok-Kun

2008-01-01

The formation of beta-carotene detergent micelles and their conversion into retinal by recombinant human beta,beta-carotene 15,15'-monooxygenase was optimized under aqueous conditions. Toluene was the most hydrophobic among the organic solvents tested; thus, it was used to dissolve beta-carotene, which is a hydrophobic compound. Tween 80 was selected as the detergent because it supported the highest level of retinal production among all of the detergents tested. The maximum production of retinal was achieved in detergent micelles containing 200 mg/L of beta-carotene and 2.4% (w/v) Tween 80. Under these conditions, the recombinant enzyme produced 97 mg/L of retinal after 16 h with a conversion yield of 48.5% (w/w). The amount of retinal produced, which is the highest ever reported, is a result of the ability of our system to dissolve large amounts of beta-carotene.

Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase.

Science.gov (United States)

Pierce, Brian C; Agger, Jane Wittrup; Wichmann, Jesper; Meyer, Anne S

2017-03-01

The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography. We demonstrate that TrCel61A is capable of oxidizing cellulose from both pretreated soy spent flake and phosphoric acid swollen cellulose, oxidizing at both the C1 and C4 positions. In addition, we show that the oxidative activity of TrCel61A displays a synergistic effect capable of boosting endoglucanase activity, and thereby substrate depolymerization of soy cellulose, by 27%. Copyright © 2016 Elsevier Inc. All rights reserved.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

OpenAIRE

Faccio Greta; Kruus Kristiina; Buchert Johanna; Saloheimo Markku

2010-01-01

Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, po...

Kynurenine 3-monooxygenase polymorphisms: relevance for kynurenic acid synthesis in patients with schizophrenia and healthy controls.

Science.gov (United States)

Holtze, Maria; Saetre, Peter; Engberg, Göran; Schwieler, Lilly; Werge, Thomas; Andreassen, Ole A; Hall, Håkan; Terenius, Lars; Agartz, Ingrid; Jönsson, Erik G; Schalling, Martin; Erhardt, Sophie

2012-01-01

Patients with schizophrenia show increased brain and cerebrospinal fluid (CSF) concentrations of the endogenous N-methyl-D-aspartate receptor antagonist kynurenic acid (KYNA). This compound is an end-metabolite of the kynurenine pathway, and its formation indirectly depends on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were selected covering KMO and were analyzed in UNPHASED. We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA. Given the limited sample size, the results are tentative until replication. Our results suggest that the nonsynonymous KMO SNP rs1053230 influences CSF concentrations of KYNA.

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene.

Science.gov (United States)

Cankar, Katarina; van Houwelingen, Adèle; Bosch, Dirk; Sonke, Theo; Bouwmeester, Harro; Beekwilder, Jules

2011-01-03

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mammalian peptidylglycine alpha-amidating monooxygenase (PAM) mRNA expression can be modulated by the La autoantigen

DEFF Research Database (Denmark)

Brenet, Fabienne; Dussault, Nadège; Borch, Jonas

2005-01-01

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated...... region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt...... downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most...

Control of cytokinin and auxin homeostasis in cyanobacteria and algae

Czech Academy of Sciences Publication Activity Database

Žižková, Eva; Kubeš, Martin; Dobrev, Petre; Přibyl, Pavel; Šimura, J.; Zahajská, Lenka; Záveská Drábková, Lenka; Novák, Ondřej; Motyka, Václav

2017-01-01

Roč. 119, č. 1 (2017), s. 151-166 ISSN 0305-7364 R&D Projects: GA ČR(CZ) GA16-14649S; GA ČR GA15-22322S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 ; RVO:67985939 Keywords : solid-phase extraction * performance liquid-chromatography * yucca flavin monooxygenases * tandem mass-spectrometry * abscisic-acid * arabidopsis-thaliana * indole-3-acetic-acid iaa * endogenous cytokinins * chlorella-vulgaris * phenylacetic acid * Cytokinin * auxin * cyanobacteria * algae * metabolism * cytokinin oxidase/dehydrogenase * cytokinin 2-methylthioderivatives * trans-zeatin * indole-3-acetic acid * tRNA Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 4.041, year: 2016

NCBI nr-aa BLAST: CBRC-DDIS-05-0051 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DDIS-05-0051 ref|YP_956349.1| Amine oxidase (flavin-containing) [Mycobacterium vanba...alenii PYR-1] gb|ABM16343.1| Amine oxidase (flavin-containing) [Mycobacterium vanbaalenii PYR-1] YP_956349.1 2e-46 36% ...

Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity.

Science.gov (United States)

Harper, Shannon M; Christie, John M; Gardner, Kevin H

2004-12-28

Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

Loss of Kynurenine 3-Mono-oxygenase Causes Proteinuria.

Science.gov (United States)

Korstanje, Ron; Deutsch, Konstantin; Bolanos-Palmieri, Patricia; Hanke, Nils; Schroder, Patricia; Staggs, Lynne; Bräsen, Jan H; Roberts, Ian S D; Sheehan, Susan; Savage, Holly; Haller, Hermann; Schiffer, Mario

2016-11-01

Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes. Copyright © 2016 by the American Society of Nephrology.

Studies of the contribution of respiratory tract metabolism to the toxicity of inhaled chemicals

International Nuclear Information System (INIS)

Dahl, A.R.; Petridou-Fischer, J.; Sabourin, P.J.; Whaley, S.; Bond, J.A.

1987-01-01

Efforts to identify specific nasal enzymes are reported. The nasal enzymes identified and characterized within this project include the cytochromes P-450, primary xenobiotic metabolizing enzymes for PAH's, nitrosamines, some halogenated compounds, alkenes, alkanes, and a large variety of other organic compounds. In addition, flavin containing monooxygenase (FMO), has been identified in the nasal cavity and characterized. Carboxylesterases, and rhodanese have also been found in the nasal cavity. Other nasal enzymes identified include: catechol methyltransferases, phenol methyltransferases, epoxide hydrolase and glutathione and glucuronyl transferases. Each of these enzyme systems may have important toxicological effects on inhaled materials. The authors have tested the ability of nasal enzymes to metabolize substrates in vivo. In these experiments, radiolabeled substances were placed on the nasal mucosas of monkeys, dogs, or Syrian hamsters. Mucus was then collected and analyzed for metabolites. It was found that benzo(a)pyrene and dihydrosafrole are both metabolized on the nasal surface following instillation by this method. Currently, experiments are planned to test the role of nasal enzymes in the fate of inhaled materials

Induction of monooxygenases and incorporation of radioactivity from 2-14C-lysine into hepatic microsomes of phenobarbital-treated rats fed a diet deficient in lysine, methionine, threonine and vitamine A, C and E

International Nuclear Information System (INIS)

Nurmagambetov, T.Zh.; Amirov, B.B.; Kuanysheva, T.K.; Sharmanov, T.Sh.

1991-01-01

The effect of diet on induction of monooxygenases and distribution of label from 2- 14 C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital (80 mg/rg, three days) was studied. 2- 14 C-lysine was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of label from 2- 14 C-lysine into fractions of liver homogenate in phenobarbital-treated rats were more pronounced as compared with the similarly trated rats that were fed a balanced diet. The possibility of mobilization of deficient essential components to liver from other organs and tissues for maintenance of monooxygenase induction is discussed

Characterization of active site residues of nitroalkane oxidase.

Science.gov (United States)

Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

2010-06-01

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. 2009 Elsevier Inc. All rights reserved.

Induction of monooxygenases and incorporation of radioactivity from 2-14C-lysine into hepatic microsomes of phenobarbital-treated rats fed a diet deficient in lysine, methionine, threonine and vitamines A, C, E

International Nuclear Information System (INIS)

Nurmagambetov, T.Zh.; Amirov, B.B.; Kuanysheva, T.G.; Sharmanov, T.Sh.

1992-01-01

The effect of diet on induction of monooxygenases and distribution of radioactivity from 2- 14 C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital was studied. 2- 14 C-lysin was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of radioactivity from 2- 14 C-lysine into fractions of liver homogenate in phenobarbital-treated rats fed diet deficient in lysine, methionine, threonine and vitamins A, C, E were more pronounced as compared with the similarly treated rats which were fed a balanced diet. The possibility of mobilization of deficient essencial components to liver from other organs and tissues for maintenance of monooxygenase induction iis discussed

Kynurenine-3-monooxygenase: a review of structure, mechanism, and inhibitors.

Science.gov (United States)

Smith, Jason R; Jamie, Joanne F; Guillemin, Gilles J

2016-02-01

Kynurenine monooxygenase (KMO) is an enzyme of the kynurenine (Kyn) pathway (KP), which is the major catabolic route of tryptophan. Kyn represents a branch point of the KP, being converted into the neurotoxin 3-hydroxykynurenine via KMO, neuroprotectant kynurenic acid, and anthranilic acid. As a result of this branch point, KMO is an attractive drug target for several neurodegenerative and/or neuroinflammatory diseases, especially Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) diseases. Although a neurological target, administration of KMO inhibitors in the periphery has demonstrated promising pharmacological results. In light of a recent crystal structure release and reports of preclinical candidates, here we provide a concise yet comprehensive update on the current state of research into the enzymology of KMO and related drug discovery efforts, highlighting areas where further work is required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of two combined genes responsible for dechlorination of 3,5,6-trichloro-2-pyridinol (TCP) in Cupriavidus pauculus P2.

Science.gov (United States)

Cao, Li; Xu, Jianhong; Wu, Guang; Li, Mingxing; Jiang, Jiandong; He, Jian; Li, Shunpeng; Hong, Qing

2013-09-15

Dehalogenation is an important mechanism for degrading and detoxifying halogenated aromatics in microbes. However, the biochemical and molecular mechanisms of dehalogenation of 3,5,6-trichloro-2-pyridinol (TCP) are still unknown. In this study, a novel 6012 bp gene cluster was cloned from TCP-degrading strain P2, which was responsible for the dehalogenation of TCP. The cluster included a monooxygenase gene (tcpA1), a flavin reductase gene (tcpB1), tcpR1, orf1 and orf2. TcpA1 and TcpB1 were indispensable for the dehalogenation of TCP. They worked together to catalyze the dehalogenation of three chlorine of TCP, and generated a more readily biodegradable product of 3,6-dihydroxypyridine-2,5-dione. TcpA1 displayed the highest activity against TCP at 40°C and at pH 8.0. Cu(2+), Zn(2+), and Hg(2+) significantly inhibited enzyme activity. To the best of our knowledge, this is the first report on a gene cluster responsible for TCP degradation. Copyright © 2013 Elsevier B.V. All rights reserved.

Spectroscopy and photophysics of flavin related compounds: Riboflavin and iso-(6,7)-riboflavin

International Nuclear Information System (INIS)

Sikorska, Ewa; Khmelinskii, Igor; Komasa, Anna; Koput, Jacek; Ferreira, Luis F.V.; Herance, Jose R.; Bourdelande, Jose L.; Williams, Sian L.; Worrall, David R.; Insinska-Rak, MaIgorzata; Sikorski, Marek

2005-01-01

The photochemistry and photophysics of isoalloxazines (10-substituted 2,3,4,10-tetrahydro-benzo[g]pteridine-2,4-diones), and especially flavins (7,8-dimethyl substituted isoalloxazines), are of considerable interest due to the biological relevance of these compounds. In this paper we report data concerning the photophysics of riboflavin and iso-(6,7)-riboflavin (10-(2,3,4,5-tetrahydroxypentyl)-6,7-dimethylbenzo[g]pteridine-2,4(3H,10H) -dione), and correlate the spectroscopic observations with theoretical calculations performed using time-dependent density functional theory. On the basis of these calculations the lowest excited singlet and triplet states are both assigned (π,π*) symmetry, and this result is used to explain the relatively low singlet oxygen quantum yields in comparison with alloxazines. Time-resolved emission studies have indicated 6,7-dimethylalloxazine a photodegradation product of iso-(6,7)-riboflavin

Cyclobutane-Containing Alkaloids: Origin, Synthesis, and Biological Activities

OpenAIRE

Sergeiko, Anastasia; Poroikov, Vladimir V; Hanuš, Lumir O; Dembitsky, Valery M

2008-01-01

Present review describes research on novel natural cyclobutane-containing alkaloids isolated from terrestrial and marine species. More than 60 biological active compounds have been confirmed to have antimicrobial, antibacterial, antitumor, and other activities. The structures, synthesis, origins, and biological activities of a selection of cyclobutane-containing alkaloids are reviewed. With the computer program PASS some additional biological activities are also predicted, which point toward ...

75 FR 26268 - Agency Information Collection Activities: Permit To Transfer Containers to a Container Station

Science.gov (United States)

2010-05-11

... Activities: Permit To Transfer Containers to a Container Station AGENCY: U.S. Customs and Border Protection... information collection requirement concerning the: Permit to Transfer Containers to a Container Station. This... information collection: Title: Permit to Transfer Containers to a Container Station. OMB Number: 1651-0049...

Transient trimethylaminuria related to menstruation

Science.gov (United States)

Shimizu, Makiko; Cashman, John R; Yamazaki, Hiroshi

2007-01-01

Background Trimethylaminuria, or fish odor syndrome, includes a transient or mild malodor caused by an excessive amount of malodorous trimethylamine as a result of body secretions. Herein, we describe data to support the proposal that menses can be an additional factor causing transient trimethylaminuria in self-reported subjects suffering from malodor and even in healthy women harboring functionally active flavin-containing monooxygenase 3 (FMO3). Methods FMO3 metabolic capacity (conversion of trimethylamine to trimethylamine N-oxide) was defined as the urinary ratio of trimethylamine N-oxide to total trimethylamine. Results Self-reported Case (A) that was homozygous for inactive Arg500stop FMO3, showed decreased metabolic capacity of FMO3 (i.e., ~10% the unaffected metabolic capacity) during 120 days of observation. For Case (B) that was homozygous for common [Glu158Lys; Glu308Gly] FMO3 polymorphisms, metabolic capacity of FMO3 was almost ~90%, except for a few days surrounding menstruation showing 90%) metabolic capacity, however, on days around menstruation the FMO3 metabolic capacity was decreased to ~60–70%. Conclusion Together, these results indicate that abnormal FMO3 capacity is caused by menstruation particularly in the presence, in homozygous form, of mild genetic variants such as [Glu158Lys; Glu308Gly] that cause a reduced FMO3 function. PMID:17257434

X-ray absorption spectroscopic studies of the dinuclear iron center in methane monooxygenase and the sulfure and chlorine centers in photographic materials

Energy Technology Data Exchange (ETDEWEB)

DeWitt, J.G.

1992-12-01

The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus and Methylosinus trichosporiwn has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(HI)Fe(IH) oxidized state of the hydroxylase from M. capsulatus consists of approximately 6 N and 0 atoms at an average distance of 2.04 [Angstrom]. The Fe-Fe distance was determined to be 3.4 [Angstrom]. No evidence for the presence of a short oxo bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MMO is significantly different from the active sites of the dinuclear iron proteins hemery and ribonucleotide reductase. In addition, the results of the first shell fits suggest that there are more oxygen than nitrogen donor ligands.

X-ray absorption spectroscopic studies of the dinuclear iron center in methane monooxygenase and the sulfure and chlorine centers in photographic materials

Energy Technology Data Exchange (ETDEWEB)

DeWitt, Jane G. [Stanford Univ., CA (United States)

1992-12-01

The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus and Methylosinus trichosporiwn has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(HI)Fe(IH) oxidized state of the hydroxylase from M. capsulatus consists of approximately 6 N and 0 atoms at an average distance of 2.04 Å. The Fe-Fe distance was determined to be 3.4 Å. No evidence for the presence of a short oxo bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MMO is significantly different from the active sites of the dinuclear iron proteins hemery and ribonucleotide reductase. In addition, the results of the first shell fits suggest that there are more oxygen than nitrogen donor ligands.

Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia.

Science.gov (United States)

Wonodi, Ikwunga; McMahon, Robert P; Krishna, Nithin; Mitchell, Braxton D; Liu, Judy; Glassman, Matthew; Hong, L Elliot; Gold, James M

2014-12-01

Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of toxic effects of Cd{sup 2+} on different fish species via liver cytochrome P450-dependent monooxygenase activities and FTIR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Henczova, Maria; Deer, Aranka Kiss [University of Szeged, Department of Biochemistry, P.O. Box 533, Szeged (Hungary); Komlosi, Viktoria [Chemical Research Center of the Hungarian Academy of Sciences, Department of Molecular Spectroscopy, P.O. Box 17, Budapest (Hungary); Mink, Janos [Chemical Research Center of the Hungarian Academy of Sciences, Department of Molecular Spectroscopy, P.O. Box 17, Budapest (Hungary); University of Veszprem, Faculty of Information Technology, Research Institute of Chemistry and Process Engineering; Analytical Chemistry Research Group of the Hungarian Academy of Sciences, P.O. Box 158, Veszprem (Hungary)

2006-06-15

The in vivo and in vitro effects of Cd{sup 2+} and the CYP1A inductor {beta}-naphthoflavone({beta}-NF) on the hepatic cytochrome P450 (Cyt 450) monooxygenases were studied in silver carp (Hypophthalmichtys molitrix V.), wels (Silurus glanis L.), and carp (Cyprinus carpio). In vivo treatment of carp with a high dose of Cd{sup 2+} (10 mg kg{sup -1}, for 3 days) caused a strong inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and a lower inhibition of 7-ethoxycoumarin-O-deethylase (ECOD) activity. The low-dose cadmium treatment (2 mg kg{sup -1} Cd{sup 2+}, for 6+3 days) resulted in 4-fold increase in EROD and a 3-fold increase in ECOD activity. The combined treatment with Cd{sup 2+} and {beta}-NF in both cases led to a loss of EROD inducibility. The silver carp and wels were treated with 10 mg L{sup -1} Cd{sup 2+} for 72 h in water. The Cyt P450 content in the wels liver microsomes was increased significantly after treatment for 48 h, whereas there was only a slight, not significant increase in Cyt P450 content in the silver carp microsomes. While the Cd{sup 2+} treatment resulted in inhibition of the CYP1A isoenzymes (EROD and ECOD), the APND (aminopyrene-N-demethylase, CYP2B or CYP3A isoenzyme) activity was increased 3- to 4-fold in both fish species. In vitro experiments of the effect of Cd{sup 2+} led to a concentration-dependent inhibition in all three investigated fish species. The ECOD isoenzyme of silver carp was the most sensitive to Cd{sup 2+}. The lowest concentration of Cd{sup 2+} resulted in 50% inhibition. The APND isoenzyme was similarly sensitive to Cd{sup 2+} in all three investigated fish species. The most sensitive species was the wels, and the least sensitive were the carp isoenzyme. FTIR spectroscopy confirmed that cadmium caused damage to the protein structure. These results support the enzyme activity measurements measured in vivo and in vitro. (orig.)

Ultra-performance liquid chromatography tandem mass-spectrometry (uplc-ms/ms) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

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A novel, rapid and sensitive Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin a...

Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

Science.gov (United States)

Johansen, Katja S

2016-02-01

The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. © 2016 Authors; published by Portland Press Limited.

Stability characteristics and structural properties of single- and double-walled boron-nitride nanotubes under physical adsorption of Flavin mononucleotide (FMN) in aqueous environment using molecular dynamics simulations

International Nuclear Information System (INIS)

Ansari, R.; Ajori, S.; Ameri, A.

2016-01-01

Graphical abstract: Structural properties and stability characteristics of single- and double-walled boron-nitride nanotubes functionalized with Flavin mononucleotide (FMN) in aqueous environment are investigated employing molecular dynamics simulations. - Highlights: • Structural and buckling analysis of boron-nitride nanotubes under physical adsorption of Flavin mononucleotide (FMN). • Gyration radius increases linearly as the weight percentage of FMN increases. • Presence of water molecules results in more expansion of FMN around BNNTs. • Critical buckling force of functionalized BNNTs is higher than that of pure BNNTs. • The critical strain of functionalized BNNTs is found to be lower than that of pure ones. - Abstract: The non-cytotoxic properties of Boron-nitride nanotubes (BNNTs) and the ability of stable interaction with biomolecules make them so promising for biological applications. In this research, molecular dynamics (MD) simulations are performed to investigate the structural properties and stability characteristics of single- and double-walled BNNTs under physical adsorption of Flavin mononucleotide (FMN) in vacuum and aqueous environments. According to the simulation results, gyration radius increases by rising the weight percentage of FMN. Also, the results demonstrate that critical buckling force of functionalized BNNTs increases in vacuum. Moreover, it is observed that by increasing the weight percentage of FMN, critical force of functionalized BNNTs rises. By contrast, critical strain reduces by functionalization of BNNTs in vacuum. Considering the aqueous environment, it is observed that gyration radius and critical buckling force of functionalized BNNTs increase more considerably than those of functionalized BNNTs in vacuum, whereas the critical strains approximately remain unchanged.

75 FR 43536 - Agency Information Collection Activities: Permit To Transfer Containers to a Container Station

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2010-07-26

... Activities: Permit To Transfer Containers to a Container Station AGENCY: U.S. Customs and Border Protection... Transfer Containers to a Container Station. This is a proposed extension of an information collection that... other technological techniques or other forms of information. Title: Permit to Transfer Containers to a...

Assessing and Modulating Kynurenine Pathway Dynamics in Huntington's Disease: Focus on Kynurenine 3-Monooxygenase.

Science.gov (United States)

Sathyasaikumar, Korrapati V; Breda, Carlo; Schwarcz, Robert; Giorgini, Flaviano

2018-01-01

The link between disturbances in kynurenine pathway (KP) metabolism and Huntington's disease (HD) pathogenesis has been explored for a number of years. Several novel genetic and pharmacological tools have recently been developed to modulate key regulatory steps in the KP such as the reaction catalyzed by the enzyme kynurenine 3-monooxygenase (KMO). This insight has offered new options for exploring the mechanistic link between this metabolic pathway and HD, and provided novel opportunities for the development of candidate drug-like compounds. Here, we present an overview of the field, focusing on some novel approaches for interrogating the pathway experimentally.

Immunoassays for riboflavin and flavin mononucleotide using antibodies specific to d-ribitol and d-ribitol-5-phosphate.

Science.gov (United States)

Ravi, G; Venkatesh, Yeldur P

2017-06-01

Riboflavin (vitamin B 2 ), a water-soluble vitamin, plays a key role in maintaining human health. Though, numerous methods have been reported for the determination of total riboflavin (TRF) content in foods and biological samples, very few methods are reported for quantifying riboflavin and its coenzymes [flavin mononucleotide (FMN); flavin adenine dinucleotide (FAD)] individually. Recently, we have demonstrated that antibodies specific to d-ribitol and d-ribitol-5-phosphate also recognize riboflavin and FMN, respectively, and not vice-versa. In this study, we have evaluated these two antibodies for the analysis of riboflavin and FMN by indirect competitive ELISA (icELISA) in selected foods and pharmaceuticals. Under the optimal assay conditions, 50% inhibition concentration (IC 50 ) and limit of detection (LOD, IC 10 ) were 3.41ng/mL and 0.02ng/mL for riboflavin, and 7.84ng/mL and 0.24ng/mL for FMN, respectively, with detectable concentration range between 0.1 and 100ng of analytes and riboflavin and FMN) from the same food samples showed variation in their values compared to TRF, and were in good agreement with values obtained from HPLC and AOAC methods. Further, spiking and recovery analysis of food samples and pharmaceuticals showed no significant matrix effects. The immunoassays were validated in terms of accuracy and precision using inter- and intra-assays. The immunoassays developed in this study are sensitive and appears feasible for screening a large number of samples in the quantification of riboflavin and FMN in various biological samples, pharmaceuticals and natural/processed foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

International Nuclear Information System (INIS)

Kwasnicka-Crawford, Dorota A.; Vincent, Steven R.

2005-01-01

Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions

Isoeugenol monooxygenase and its putative regulatory gene are located in the eugenol metabolic gene cluster in Pseudomonas nitroreducens Jin1.

Science.gov (United States)

Ryu, Ji-Young; Seo, Jiyoung; Unno, Tatsuya; Ahn, Joong-Hoon; Yan, Tao; Sadowsky, Michael J; Hur, Hor-Gil

2010-03-01

The plant-derived phenylpropanoids eugenol and isoeugenol have been proposed as useful precursors for the production of natural vanillin. Genes involved in the metabolism of eugenol and isoeugenol were clustered in region of about a 30 kb of Pseudomonas nitroreducens Jin1. Two of the 23 ORFs in this region, ORFs 26 (iemR) and 27 (iem), were predicted to be involved in the conversion of isoeugenol to vanillin. The deduced amino acid sequence of isoeugenol monooxygenase (Iem) of strain Jin1 had 81.4% identity to isoeugenol monooxygenase from Pseudomonas putida IE27, which also transforms isoeugenol to vanillin. Iem was expressed in E. coli BL21(DE3) and was found to lead to isoeugenol to vanillin transformation. Deletion and cloning analyses indicated that the gene iemR, located upstream of iem, is required for expression of iem in the presence of isoeugenol, suggesting it to be the iem regulatory gene. Reverse transcription, real-time PCR analyses indicated that the genes involved in the metabolism of eugenol and isoeugenol were differently induced by isoeugenol, eugenol, and vanillin.

Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

Science.gov (United States)

Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

2017-04-15

Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effect of NGATHA altered activity in auxin signaling pathways within the Arabidopsis gynoecium

Directory of Open Access Journals (Sweden)

Irene eMartinez-Fernandez

2014-05-01

Full Text Available The four NGATHA genes (NGA form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium.We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors.

Active containment systems incorporating modified pillared clays

International Nuclear Information System (INIS)

Lundie, P.; McLeod, N.

1997-01-01

The application of treatment technologies in active containment systems provides a more advanced and effective method for the remediation of contaminated sites. These treatment technologies can be applied in permeable reactive walls and/or funnel and gate systems. The application of modified pillared clays in active containment systems provides a mechanism for producing permeable reactive walls with versatile properties. These pillared clays are suitably modified to incorporate reactive intercalatants capable of reacting with both a broad range of organic pollutants of varying molecular size, polarity and reactivity. Heavy metals can be removed from contaminated water by conventional ion-exchange and other reactive processes within the clay structure. Complex contamination problems can be addressed by the application of more than one modified clay on a site specific basis. This paper briefly describes the active containment system and the structure/chemistry of the modified pillared clay technology, illustrating potential applications of the in-situ treatment process for contaminated site remediation

Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker [3H]GBR-12935

International Nuclear Information System (INIS)

Kalow, W.; Tyndale, R.F.; Niznik, H.B.; Inaba, T.

1990-01-01

P450IID6 (human sparteine monooxygenase) metabolizes many drugs including neuroleptics, antidepressants, and beta-blockers. The P450IID6 exists in human, bovine, rat and canine brains, but in very low quantities causing methodological difficulties in its assessment. Work with [ 3 H]GBR-12935; 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenyl propyl) piperazine has shown that it binds a neuronal/hepatic protein with high affinity (∼7nM) and a rank order of inhibitory potency suggesting that the binding protein is cytochrome P450IID6. The binding was used to predict that d-amphetamine and methamphetamine would interact with P450IID6. Inhibition studies indicated that these compounds were competitive inhibitors of P450IID6. Haloperidol (HAL) and it's metabolite hydroxy-haloperidol (RHAL) are both competitive inhibitors of P450IID6 activity and were found to inhibit [ 3 H]GBR-12935 binding. K i values of twelve compounds (known to interact with the DA transporter or P450IID6) for [ 3 H]GRB-12935 binding and P450IID6 activity. The techniques are now available for measurements of cytochrome P450IID6 in healthy and diseased brain/liver tissue using radio-receptor binding assay techniques with [ 3 H]GBR-12935

Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

Science.gov (United States)

Minerdi, Daniela; Zgrablic, Ivan; Castrignanò, Silvia; Catucci, Gianluca; Medana, Claudio; Terlizzi, Maria Elena; Gribaudo, Giorgio; Gilardi, Gianfranco; Sadeghi, Sheila J

2016-01-01

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

Science.gov (United States)

Weber, Heike E; Gottardi, Manuela; Brückner, Christine; Oreb, Mislav; Boles, Eckhard; Tripp, Joanna

2017-05-15

Biotechnological production of cis , cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis , cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis , cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C iso (AroY-C iso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1 , which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-C iso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis , cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

Science.gov (United States)

Wille, Georg; Ritter, Michaela; Weiss, Manfred S; König, Stephan; Mäntele, Werner; Hübner, Gerhard

2005-04-05

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

International Nuclear Information System (INIS)

Atia, K.S.

2005-01-01

The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

Study on preventive effects of i.v. administration of flavin adenine dinucleotide (FAD) before irradiation on radiation stomatitis

International Nuclear Information System (INIS)

Nagai, Masao; Houzawa, Jiro; Hakamada, Masaru

1984-01-01

In order to compare the preventive effect on radiation stomatitis, flavin adenine dinucleotide (FAD) or vitamin C was administered intravenously until the blood level reached the maximum at the time of irradiation. Thirtyfive patients with cranial or cervical tumors were allocated into the group with FAD (15), the group with vitamin C (10), and the group with irradiation alone (10). The incidence of stomititis was significantly lower and the number of patients in whom the drug was withdrawn due to stomatitis was extremely smaller in the group with FAD than in the other groups. FAD administered before irradiation was considered very useful in preventing radiation stomatitis. (Namekawa, K.)

Noncanonical Reactions of Flavoenzymes

Directory of Open Access Journals (Sweden)

Pablo Sobrado

2012-11-01

Full Text Available Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a “molecular scaffold” in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates.

YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana

KAUST Repository

Kim, Jeong Im

2011-04-21

The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes. 2011 The Author(s).

Coenzyme Recognition and Gene Regulation by a Flavin Mononucleotide Riboswitch

Energy Technology Data Exchange (ETDEWEB)

Serganov, A.; Huang, L; Patel, D

2009-01-01

The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg{sup 2+}-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

Effect of two-linked mutations of the FMO3 gene on itopride metabolism in Chinese healthy volunteers.

Science.gov (United States)

Zhou, Li-Ping; Tan, Zhi-Rong; Chen, Hao; Guo, Dong; Chen, Yao; Huang, Wei-Hua; Wang, Lian-Sheng; Zhang, Guo-Gang

2014-11-01

Itopride is an effective gastroprokinetic agent mainly used for the treatment of functional dyspepsia. Flavin-containing monooxygenase 3 (FMO3) has been confirmed to be the key enzyme involved in the main itopride metabolic pathway. We investigated whether the FMO3 genotypes can affect itopride metabolism in Chinese healthy volunteers. Twelve healthy volunteers who had been genotyped for FMO3 gene were selected to participate in our study. Volunteers were given 50 mg itopride orally and then blood samples were collected from 0 to 24 h. The plasma concentrations of itopride and itopride N-oxide were determined by HPLC-MS/MS method. Itopride and itopride N-oxide both exhibit FMO3 genotype-dependent pharmacokinetic profiles. The area under the plasma concentration-time curve (AUC) of itopride increased by 127.82 ± 41.99 % (P itopride N-oxide decreased by 30.30 ± 25.70 % (P itopride and itopride N-oxide were observed between these two genotypes. The FMO3 allele can significantly affect the metabolism of itopride. The pharmacokinetic parameters of both itopride and itopride N-oxide were significantly different between these two genotypes.

Structural and molecular dynamics studies of a C1-oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

Energy Technology Data Exchange (ETDEWEB)

Liu, Bing [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Kognole, Abhishek A. [Department of Chemical and Materials Engineering, University of Kentucky, Lexington KY USA; Wu, Miao [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Westereng, Bjørge [Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås Norway; Crowley, Michael F. [Biosciences Center, National Renewable Energy Laboratory, Golden CO USA; Kim, Seonah [Biosciences Center, National Renewable Energy Laboratory, Golden CO USA; Dimarogona, Maria [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Department of Chemical Engineering, University of Patras, Greece; Payne, Christina M. [Department of Chemical and Materials Engineering, University of Kentucky, Lexington KY USA; Directorate of Engineering, Division of Chemical, Bioengineering, Environmental, and Transport Systems, National Science Foundation, Alexandria VA USA; Sandgren, Mats [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden

2018-04-24

Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white-rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 A resolution using X-ray crystallography. Unlike the majority of the currently available C1-specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting ..beta..-strands ..beta..2 and ..beta..3 of the ..beta..-sandwich structure. Molecular dynamics (MD) simulations suggest roles for both aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active-site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1-specific LPMOs, giving a molecular-level picture of active site substrate interactions.

Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

Science.gov (United States)

Stephens, Geoffrey L; Wang, Qun; Swerdlow, Bonnie; Bhat, Geetha; Kolbeck, Roland; Fung, Michael

2013-07-01

The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia✰,✰✰

Science.gov (United States)

Wonodi, Ikwunga; McMahon, Robert P.; Krishna, Nithin; Mitchell, Braxton D.; Liu, Judy; Glassman, Matthew; Hong, L. Elliot; Gold, James M.

2015-01-01

Background Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. Methods We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Results Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. Conclusions These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. PMID:25464917

Physical map location of the multicopy genes coding for ammonia monooxygenase and hydroxylamine oxidoreductase in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

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Hirota, R; Yamagata, A; Kato, J; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H

2000-02-01

Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB(1) and amoCAB(2)), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)). In this DNA fragment, amoCAB(1) and amoCAB(2) were about 390 kb apart, while hao(1), hao(2), and hao(3) were separated by at least about 100 kb from each other. Interestingly, hao(1) and hao(2) were located relatively close to amoCAB(1) and amoCAB(2), respectively. DNA sequence analysis revealed that hao(1) and hao(2) shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao(3) showed only 30% nucleotide identity in the 160-bp corresponding region.

Cloning of genes related to aliphatic glucosinolate metabolism and the mechanism of sulforaphane accumulation in broccoli sprouts under jasmonic acid treatment.

Science.gov (United States)

Guo, Liping; Yang, Runqiang; Gu, Zhenxin

2016-10-01

Cytochrome P450 79F1 (CYP79F1), cytochrome P450 83A1 (CYP83A1), UDP-glucosyltransferase 74B1 (UGT74B1), sulfotransferase 18 (ST5b) and flavin-containing monooxygenase GS-OX1 (FMOGS - OX1 ) are important enzymes in aliphatic glucosinolate biosynthesis. In this study, their full-length cDNA in broccoli was firstly cloned, then the mechanism of sulforaphane accumulation under jasmonic acid (JA) treatment was investigated. The full-length cDNA of CYP79F1, CYP83A1, UGT74B1, ST5b and FMOGS - OX1 comprised 1980, 1652, 1592, 1378 and 1623 bp respectively. The increase in aliphatic glucosinolate accumulation in broccoli sprouts treated with JA was associated with elevated expression of genes in the aliphatic glucosinolate biosynthetic pathway. Application of 100 µmol L(-1) JA increased myrosinase (MYR) activity but did not affect epithiospecifier protein (ESP) activity in broccoli sprouts, which was supported by the expression of MYR and ESP. Sulforaphane formation in 7-day-old sprouts treated with 100 µmol L(-1) JA was 3.36 and 1.30 times that in the control and 300 µmol L(-1) JA treatment respectively. JA enhanced the accumulation of aliphatic glucosinolates in broccoli sprouts via up-regulation of related gene expression. Broccoli sprouts treated with 100 µmol L(-1) JA showed higher sulforphane formation than those treated with 300 µmol L(-1) JA owing to the higher glucoraphanin content and myrosinase activity under 100 µmol L(-1) JA treatment. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Importance of kynurenine 3-monooxygenase for spontaneous firing and pharmacological responses of midbrain dopamine neurons: Relevance for schizophrenia.

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Tufvesson-Alm, Maximilian; Schwieler, Lilly; Schwarcz, Robert; Goiny, Michel; Erhardt, Sophie; Engberg, Göran

2018-06-05

Kynurenine 3-monooxygenase (KMO) is an essential enzyme of the kynurenine pathway, converting kynurenine into 3-hydroxykynurenine. Inhibition of KMO increases kynurenine, resulting in elevated levels of kynurenic acid (KYNA), an endogenous N-methyl-d-aspartate and α*7-nicotinic receptor antagonist. The concentration of KYNA is elevated in the brain of patients with schizophrenia, possibly as a result of a reduced KMO activity. In the present study, using in vivo single cell recording techniques, we investigated the electrophysiological characteristics of ventral tegmental area dopamine (VTA DA) neurons and their response to antipsychotic drugs in a KMO knock-out (K/O) mouse model. KMO K/O mice exhibited a marked increase in spontaneous VTA DA neuron activity as compared to wild-type (WT) mice. Furthermore, VTA DA neurons showed clear-cut, yet qualitatively opposite, responses to the antipsychotic drugs haloperidol and clozapine in the two genotypes. The anti-inflammatory drug parecoxib successfully lowered the firing activity of VTA DA neurons in KMO K/O, but not in WT mice. Minocycline, an antibiotic and anti-inflammatory drug, produced no effect in this regard. Taken together, the present data further support the usefulness of KMO K/O mice for studying distinct aspects of the pathophysiology and pharmacological treatment of psychiatric disorders such as schizophrenia. Copyright © 2018. Published by Elsevier Ltd.

Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.

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Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

2016-04-14

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones

DEFF Research Database (Denmark)

Bauer, M; Suppmann, S; Meyer, M

2002-01-01

in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting...... by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase...... that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that GDNF, in addition to its action in structural differentiation, also promotes differentiation regarding expression and enzymatic activity of a crucial component...

Monooxygenase system in Guerin’s carcinoma of rats under conditions of ω-3 polyunsaturated fatty acids administration

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M. M. Marchenko

2016-08-01

Full Text Available The aim of the study was to determine the variations of function in components of monooxygenase system (MOS of rat Guerin’s carcinoma under ω-3 polyunsaturated fatty acids (PUFAs administration. The activity of Guerin’s carcinoma microsomal NADH-cytochrome b5 reductase, the content and the rate of cytochrome b5 oxidation-reduction, the content and the rate of cytochrome Р450 oxidation-reduction have been investigated in rats with tumor under conditions of ω-3 PUFAs administration. ω-3 PUFAs supplementation before and after transplantation of Guerin’s carcinoma resulted in the increase of NADH-cytochrome b5 reductase activity and decrease of cytochrome b5 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. Increased activity of NADH-cytochrome b5 reductase facilitates higher electron flow in redox-chain of MOS. Under decreased cytochrome b5 levels the electrons are transferred to oxygen, which leads to heightened generation of superoxide (O2•- in comparison to control. It was shown, that the decrease of cytochrome P450 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of oncogenesis under ω-3 PUFAs administration may be associated with its transition into an inactive form – cytochrome P420. This decrease in cytochrome P450 coincides with increased generation of superoxide by MOS oxygenase chain.

Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

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Nagayoshi, Haruna; Kakimoto, Kensaku; Konishi, Yoshimasa; Kajimura, Keiji; Nakano, Takeshi

2017-10-17

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

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Molnár, István; Hill, D. Steven; Zirkle, Ross; Hammer, Philip E.; Gross, Frank; Buckel, Thomas G.; Jungmann, Volker; Pachlatko, Johannes Paul; Ligon, James M.

2005-01-01

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. PMID:16269733

Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

DEFF Research Database (Denmark)

Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

2014-01-01

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence....... In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important...

Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

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Jain, Chakresh Kumar; Gupta, Money; Prasad, Yamuna; Wadhwa, Gulshan; Sharma, Sanjeev Kumar

2014-07-01

The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

From Cholesterogenesis to Steroidogenesis: Role of Riboflavin and Flavoenzymes in the Biosynthesis of Vitamin D12

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Pinto, John T.; Cooper, Arthur J. L.

2014-01-01

Flavin-dependent monooxygenases and oxidoreductases are located at critical branch points in the biosynthesis and metabolism of cholesterol and vitamin D. These flavoproteins function as obligatory intermediates that accept 2 electrons from NAD(P)H with subsequent 1-electron transfers to a variety of cytochrome P450 (CYP) heme proteins within the mitochondria matrix (type I) and the (microsomal) endoplasmic reticulum (type II). The mode of electron transfer in these systems differs slightly in the number and form of the flavin prosthetic moiety. In the type I mitochondrial system, FAD-adrenodoxin reductase interfaces with adrenodoxin before electron transfer to CYP heme proteins. In the microsomal type II system, a diflavin (FAD/FMN)-dependent cytochrome P450 oxidoreductase [NAD(P)H-cytochrome P450 reductase (CPR)] donates electrons to a multitude of heme oxygenases. Both flavoenzyme complexes exhibit a commonality of function with all CYP enzymes and are crucial for maintaining a balance of cholesterol and vitamin D metabolites. Deficits in riboflavin availability, imbalances in the intracellular ratio of FAD to FMN, and mutations that affect flavin binding domains and/or interactions with client proteins result in marked structural alterations within the skeletal and central nervous systems similar to those of disorders (inborn errors) in the biosynthetic pathways that lead to cholesterol, steroid hormones, and vitamin D and their metabolites. Studies of riboflavin deficiency during embryonic development demonstrate congenital malformations similar to those associated with genetic alterations of the flavoenzymes in these pathways. Overall, a deeper understanding of the role of riboflavin in these pathways may prove essential to targeted therapeutic designs aimed at cholesterol and vitamin D metabolism. PMID:24618756

Intermittent but not sustained hypoxia activates orexin-containing neurons in mice.

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Yamaguchi, Keiji; Futatsuki, Takahiro; Ushikai, Jumpei; Kuroki, Chiharu; Minami, Toshiaki; Kakihana, Yasuyuki; Kuwaki, Tomoyuki

2015-01-15

Hypothalamic orexin-containing neurons are activated by CO2 and contribute to hypercapnic ventilatory activation. However, their role in oxygen-related regulation of breathing is not well defined. In this study, we examined whether an experimental model mimicking apnea-induced repetitive hypoxemia (intermittent hypoxia [IH]) activates orexin-containing neurons. Mice were exposed to IH (5×5min at 10% O2), intermittent hyperoxia (IO; 5×5min at 50% O2), sustained hypoxia (SH; 25min at 10% O2), or sham stimulation. Their brains were examined using double immunohistochemical staining for orexin and c-Fos. The results indicated that IH (25.8±3.0%), but not SH (9.0±1.5%) activated orexin-containing neurons when compared to IO (5.5±0.6%) and sham stimulation (5.9±1.4%). These results correlate with those of our previous work showing that IH-induced respiratory long-term facilitation is dependent on orexin-containing neurons. Taken together, orexin contributes to repetitive hypoxia-induced respiratory activation and the hypoxic activation of orexin-containing neurons is pattern dependent. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

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Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

2015-10-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

Science.gov (United States)

Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

2013-07-01

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

Science.gov (United States)

Hussain, Razak; Kumari, Indu; Sharma, Shikha; Ahmed, Mushtaq; Khan, Tabreiz Ahmad; Akhter, Yusuf

2017-12-01

Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

Xenobiotica-metabolizing enzymes in the skin of rat, mouse, pig, guinea pig, man, and in human skin models.

Science.gov (United States)

Oesch, F; Fabian, E; Landsiedel, Robert

2018-06-18

Studies on the metabolic fate of medical drugs, skin care products, cosmetics and other chemicals intentionally or accidently applied to the human skin have become increasingly important in order to ascertain pharmacological effectiveness and to avoid toxicities. The use of freshly excised human skin for experimental investigations meets with ethical and practical limitations. Hence information on xenobiotic-metabolizing enzymes (XME) in the experimental systems available for pertinent studies compared with native human skin has become crucial. This review collects available information of which-taken with great caution because of the still very limited data-the most salient points are: in the skin of all animal species and skin-derived in vitro systems considered in this review cytochrome P450 (CYP)-dependent monooxygenase activities (largely responsible for initiating xenobiotica metabolism in the organ which provides most of the xenobiotica metabolism of the mammalian organism, the liver) are very low to undetectable. Quite likely other oxidative enzymes [e.g. flavin monooxygenase, COX (cooxidation by prostaglandin synthase)] will turn out to be much more important for the oxidative xenobiotic metabolism in the skin. Moreover, conjugating enzyme activities such as glutathione transferases and glucuronosyltransferases are much higher than the oxidative CYP activities. Since these conjugating enzymes are predominantly detoxifying, the skin appears to be predominantly protected against CYP-generated reactive metabolites. The following recommendations for the use of experimental animal species or human skin in vitro models may tentatively be derived from the information available to date: for dermal absorption and for skin irritation esterase activity is of special importance which in pig skin, some human cell lines and reconstructed skin models appears reasonably close to native human skin. With respect to genotoxicity and sensitization reactive

Reductive Dissolution of Goethite and Hematite by Reduced Flavins

Energy Technology Data Exchange (ETDEWEB)

Shi, Zhi; Zachara, John M.; Wang, Zheming; Shi, Liang; Fredrickson, Jim K.

2013-10-02

The abiotic reductive dissolution of goethite and hematite by the reduced forms of flavin mononucleotide (FMNH2) and riboflavin (RBFH2), electron transfer mediators (ETM) secreted by the dissimilatory iron-reducing bacterium Shewanella, was investigated under stringent anaerobic conditions. In contrast to the rapid redox reaction rate observed for ferrihydrite and lepidocrocite (Shi et al., 2012), the reductive dissolution of crystalline goethite and hematite was slower, with the extent of reaction limited by the thermodynamic driving force at circumneutral pH. Both the initial reaction rate and reaction extent increased with decreasing pH. On a unit surface area basis, goethite was less reactive than hematite between pH 4.0 and 7.0. AH2DS, the reduced form of the well-studied synthetic ETM anthraquinone-2,6-disulfonate (AQDS), yielded higher rates than FMNH2 under most reaction conditions, despite the fact that FMNH2 was a more effective reductant than AH2DS for ferryhydrite and lepidocrocite. Two additional model compounds, methyl viologen and benzyl viologen, were investigated under similar reaction conditions to explore the relationship between reaction rate and thermodynamic properties. Relevant kinetic data from the literature were also included in the analysis to span a broad range of half-cell potentials. Other conditions being equal, the surface area normalized initial reaction rate (ra) increased as the redox potential of the reductant became more negative. A non-linear, parabolic relationship was observed between log ra and the redox potential for eight reducants at pH 7.0, as predicted by Marcus theory for electron transfer. When pH and reductant concentration were fixed, log ra was positively correlated to the redox potential of four Fe(III) oxides over a wide pH range, following a non-linear parabolic relationship as well.

Kynurenine–3–monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis

Science.gov (United States)

Mole, Damian J; Webster, Scott P; Uings, Iain; Zheng, Xiaozhong; Binnie, Margaret; Wilson, Kris; Hutchinson, Jonathan P; Mirguet, Olivier; Walker, Ann; Beaufils, Benjamin; Ancellin, Nicolas; Trottet, Lionel; Bénéton, Véronique; Mowat, Christopher G; Wilkinson, Martin; Rowland, Paul; Haslam, Carl; McBride, Andrew; Homer, Natalie ZM; Baily, James E; Sharp, Matthew GF; Garden, O James; Hughes, Jeremy; Howie, Sarah EM; Holmes, Duncan S; Liddle, John; Iredale, John P

2015-01-01

Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death1,2 Acute mortality from AP-MODS exceeds 20%3 and for those who survive the initial episode, their lifespan is typically shorter than the general population4. There are no specific therapies available that protect individuals against AP-MODS. Here, we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism5, is central to the pathogenesis of AP-MODS. We created a mouse strain deficient for Kmo with a robust biochemical phenotype that protected against extrapancreatic tissue injury to lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in levels of kynurenine pathway metabolites in vivo and afforded therapeutic protection against AP-MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS and open up a new area for drug discovery in critical illness. PMID:26752518

MICAL, the Flavoenzyme Participating in Cytoskeleton Dynamics

Directory of Open Access Journals (Sweden)

Daniela Zucchini

2013-03-01

Full Text Available MICAL (from the Molecule Interacting with CasL indicates a family of recently discovered cytosolic, multidomain proteins, which uniquely couple an N-terminal FAD-containing monooxygenase-like domain to typical calponine homology, LIM and coiled-coil protein-interaction modules. Genetic and cell biology approaches have demonstrated an essential role of the catalytic activity of the monooxygenase-like domain in transducing the signal initiated by semaphorins interaction with their plexin receptors, which results in local actin cytoskeleton disassembly as part of fundamental processes that include differentiation, migration and cell-cell contacts in neuronal and non-neuronal cell types. This review focuses on the structure-function relations of the MICAL monooxygenase-like domain as they are emerging from the available in vitro studies on mouse, human and Drosophila MICAL forms that demonstrated a NADPH-dependent actin depolymerizing activity of MICAL. With Drosophila MICAL forms, actin depolymerization was demonstrated to be associated to conversion of Met44 to methionine sulfone through a postulated hydroxylating reaction. Arguments supporting the concept that MICAL effect on F-actin may be reversible will be discussed.

Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis.

Science.gov (United States)

Mole, Damian J; Webster, Scott P; Uings, Iain; Zheng, Xiaozhong; Binnie, Margaret; Wilson, Kris; Hutchinson, Jonathan P; Mirguet, Olivier; Walker, Ann; Beaufils, Benjamin; Ancellin, Nicolas; Trottet, Lionel; Bénéton, Véronique; Mowat, Christopher G; Wilkinson, Martin; Rowland, Paul; Haslam, Carl; McBride, Andrew; Homer, Natalie Z M; Baily, James E; Sharp, Matthew G F; Garden, O James; Hughes, Jeremy; Howie, Sarah E M; Holmes, Duncan S; Liddle, John; Iredale, John P

2016-02-01

Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.

Screening of Bothrops snake venoms for L-amino acid oxidase activity

Energy Technology Data Exchange (ETDEWEB)

Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F. [Federal Univ. of Parana, Curitiba (Brazil)

1995-12-31

Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

Antimicrobial activity and pH of a endodontic sealer containing MTA

DEFF Research Database (Denmark)

Maliza, Amanda GA; de Andrade, Flaviana Bombarda; Arias, Marcela C

Objective: To investigate the antimicrobial activity, calcium release, and pH of a new mineral trioxide aggregate endodontic sealer when compared to endodontic sealers containing calcium hydroxide and/or epoxy resin. Method: Specimens were fabricated from MTA Fillapex, Sealer 26, Sealapex, and AH...... Plus immediately, 24 or 48 hours prior to the tests. The antimicrobial activity against Enterococcus faecalis and Candida albicans was evaluated by the direct contact and the agar diffusion methods. Calcium release was determined by atomic absorption spectrometry. The pH from solutions containing...... in solution with an alkaline pH. Conclusion: The new mineral trioxide aggregate endodontic sealer presented higher antimicrobial activity when compared to the sealers containing calcium hydroxide and/or epoxy resin. As for pH and calcium release, the sealers containing calcium hydroxide resulted in presented...

Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor.

Science.gov (United States)

Vidal, Juan-C; Espuelas, Javier; Castillo, Juan-R

2004-10-01

A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.

In planta functions of cytochrome P450 monooxygenase genes in the phytocassane biosynthetic gene cluster on rice chromosome 2.

Science.gov (United States)

Ye, Zhongfeng; Yamazaki, Kohei; Minoda, Hiromi; Miyamoto, Koji; Miyazaki, Sho; Kawaide, Hiroshi; Yajima, Arata; Nojiri, Hideaki; Yamane, Hisakazu; Okada, Kazunori

2018-06-01

In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta.

Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

Science.gov (United States)

Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

2012-11-01

Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Synthesis and Insecticidal Activities of Novel Analogues of Chlorantraniliprole Containing Nitro Group

Institute of Scientific and Technical Information of China (English)

FENG Qi; WANG Ming-zhong; XIONG Li-xia; LIU Zhi-li; LI Zheng-ming

2011-01-01

Twelve novel analogues of chlorantraniliprole containing nitro group were synthesized,and their structures were characterized by 1H NMR and high-resolution mass spectrometry(HRMS).Their evaluated insecticidal activities against oriental armyworm(Mythimna separata) indicate that the nitro-containing analogues showed favorable insecticidal activities,while the activity of compounds 5g at 0.25 mg/L was 40%,but still lower than chlorantraniliprole.

Effect of radio-detoxified endotoxin on the liver microsomal drug metabolizing enzyme system in rats

International Nuclear Information System (INIS)

Bertok, L.; Szeberenyi, S.

1983-01-01

E. coli endotoxin (LPS) depresses the hepatic microsomal mono-oxygenase activity. Radio-detoxified LPS (TOLERIN: 60 Co irradiated endotoxin preparation) decreases this biotransforming activity to a smaller extent. Phenobarbital, an inducer of this mono-oxygenase system, failed to induce in LPS-treated animals. In radio-detoxified LPS-treated rats, phenobarbital induced the mono-oxygenase and almost fully restored the biotransformation

Expression and purification of the metal-containing monooxygenases tryptophan hydroxylase and dopamine β-hydroxylase

DEFF Research Database (Denmark)

Karlsen, Pernille Efferbach

-hyperactive disorder (ADHD) among others. Since all these diseases are the cause of huge economical and personal costs it is very important to gain more knowledge of TPH and DβH since these two enzymes could be possible targets for medicine against the diseases mentioned above. TPH a three-domain, iron......-containing enzyme which belongs to the aromatic amino acid hydroxylase (AAAH) family. It exist in two isoforms, TPH1 and TPH2, which are expressed in different tissues and have different properties. TPH is known as a very diffcult protein to work with especially due to instability and only truncated forms of TPH1...... have been purified and crystallized. This project concern the human neuronal TPH or TPH2. In an attempt to overcome the problems with recombinant TPH two stability and solubility optimized variants of TPH2 are designed. Escherichia coli (E. coli) expression strains for these variants and full length...

ELONGATED UPPERMOST INTERNODE Encodes a Cytochrome P450 Monooxygenase That Epoxidizes Gibberellins in a Novel Deactivation Reaction in RiceW⃞

Science.gov (United States)

Zhu, Yongyou; Nomura, Takahito; Xu, Yonghan; Zhang, Yingying; Peng, Yu; Mao, Bizeng; Hanada, Atsushi; Zhou, Haicheng; Wang, Renxiao; Li, Peijin; Zhu, Xudong; Mander, Lewis N.; Kamiya, Yuji; Yamaguchi, Shinjiro; He, Zuhua

2006-01-01

The recessive tall rice (Oryza sativa) mutant elongated uppermost internode (eui) is morphologically normal until its final internode elongates drastically at the heading stage. The stage-specific developmental effect of the eui mutation has been used in the breeding of hybrid rice to improve the performance of heading in male sterile cultivars. We found that the eui mutant accumulated exceptionally large amounts of biologically active gibberellins (GAs) in the uppermost internode. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase, CYP714D1. Using heterologous expression in yeast, we found that EUI catalyzed 16α,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall and dwarfed phenotypes of the eui mutant and Eui-overexpressing transgenic plants, respectively, 16α,17-epoxidation reduced the biological activity of GA4 in rice, demonstrating that EUI functions as a GA-deactivating enzyme. Expression of Eui appeared tightly regulated during plant development, in agreement with the stage-specific eui phenotypes. These results indicate the existence of an unrecognized pathway for GA deactivation by EUI during the growth of wild-type internodes. The identification of Eui as a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops. PMID:16399803

The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea

Directory of Open Access Journals (Sweden)

Kang Zhang

2018-05-01

Full Text Available A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO, was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO were similar to those of the wild-type (WT strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP and mitogen-activated protein kinase (MAPK signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea.

The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea.

Science.gov (United States)

Zhang, Kang; Yuan, Xuemei; Zang, Jinping; Wang, Min; Zhao, Fuxin; Li, Peifen; Cao, Hongzhe; Han, Jianmin; Xing, Jihong; Dong, Jingao

2018-01-01

A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea . A novel pathogenicity-related gene BcKMO , which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO -complementing mutant (BCG183/ BcKMO ) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/ BcKMO . Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H 2 O 2 , and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1 , Pka2 , PkaR , Bcg2 , Bcg3 , bmp1 , and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea . Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea .

Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

Directory of Open Access Journals (Sweden)

Peter K Busk

Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine.

Science.gov (United States)

Han, Bing; Chen, Yin; Abell, Guy; Jiang, Hao; Bodrossy, Levente; Zhao, Jiangang; Murrell, J Colin; Xing, Xin-Hui

2009-11-01

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus/Methylocystis, type I methanotrophs related to Methylobacter/Methylosoma and Methylococcus, and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using (13)CH(4) were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella, which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium, were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Energy Technology Data Exchange (ETDEWEB)

Han, Bing; Chen, Yin; Abell, Guy; Jiang, Hao; Bodrossy, Levente; Zhao, Jiangang; Murrell, J. Colin; Xing, Xin-Hui [Tsinghua University, Beijing (China). Dept. of Chemical Engineering

2009-11-15

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus/Methylocystis, type I methanotrophs related to Methylobacter/Methylosoma and Methylococcus, and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using {sup 13}CH{sub 4} were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella, which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium, were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.

Identification of active methanotrophs in a landfill cover soil through detection of expression of 16S rRNA and functional genes.

Science.gov (United States)

Chen, Yin; Dumont, Marc G; Cébron, Aurélie; Murrell, J Colin

2007-11-01

Active methanotrophs in a landfill soil were revealed by detecting the 16S rRNA of methanotrophs and the mRNA transcripts of key genes involved in methane oxidation. New 16S rRNA primers targeting type I and type II methanotrophs were designed and optimized for analysis by denaturing gradient gel electrophoresis. Direct extraction of RNA from soil enabled the analysis of the expression of the functional genes: mmoX, pmoA and mxaF, which encode subunits of soluble methane monooxygenase, particulate methane monooxygenase and methanol dehydrogenase respectively. The 16S rRNA polymerase chain reaction (PCR) primers for type I methanotrophs detected Methylomonas, Methylosarcina and Methylobacter sequences from both soil DNA and cDNA which was generated from RNA extracted directly from the landfill cover soil. The 16S rRNA primers for type II methanotrophs detected primarily Methylocella and some Methylocystis 16S rRNA genes. Phylogenetic analysis of mRNA recovered from the soil indicated that Methylobacter, Methylosarcina, Methylomonas, Methylocystis and Methylocella were actively expressing genes involved in methane and methanol oxidation. Transcripts of pmoA but not mmoX were readily detected by reverse transcription polymerase chain reaction (RT-PCR), indicating that particulate methane monooxygenase may be largely responsible for methane oxidation in situ.

Two Arabidopsis cytochrome P450 monooxygenases, CYP714A1 and CYP714A2, function redundantly in plant development through gibberellin deactivation.

Science.gov (United States)

Zhang, Yingying; Zhang, Baichen; Yan, Dawei; Dong, Weixin; Yang, Weibing; Li, Qun; Zeng, Longjun; Wang, Jianjun; Wang, Linyou; Hicks, Leslie M; He, Zuhua

2011-07-01

The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA(12) slightly accumulated in the transgenic rice, and GA(19) highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Acoustically active lipospheres containing paclitaxel: a new therapeutic ultrasound contrast agent.

Science.gov (United States)

Unger, E C; McCreery, T P; Sweitzer, R H; Caldwell, V E; Wu, Y

1998-12-01

Paclitaxel-carrying lipospheres (MRX-552) were developed and evaluated as a new ultrasound contrast agent for chemotherapeutic drug delivery. Paclitaxel was suspended in soybean oil and added to an aqueous suspension of phospholipids in vials. The headspace of the vials was replaced with perfluorobutane gas; the vials were sealed, and they were agitated at 4200 rpm on a shaking device. The resulting lipospheres containing paclitaxel were studied for concentration, size, acute toxicity in mice, and acoustic activity and drug release with ultrasound. Lipospheres containing sudan black dye were produced to demonstrate the acoustically active liposphere (AAL)-ultrasound release concept. Acoustically active lipospheres containing paclitaxel had a mean particle count of approximately 1 x 10(9) particles per mL and a mean size of 2.9 microns. Acute toxicity studies in mice showed a 10-fold reduction in toxicity for paclitaxel in AALs compared with free paclitaxel. The AALs reflected ultrasound as a contrast agent. Increasing amounts of ultrasound energy selectively ruptured the AALs and released the paclitaxel. Acoustically active lipospheres represent a new class of acoustically active drug delivery vehicles. Future studies will assess efficacy of AALs for ultrasound-mediated drug delivery.

Antitumor Active Protein-containing Glycans from the Body of Ganoderma tsugae

Institute of Scientific and Technical Information of China (English)

LIU Ying; LI Yue-fei; ZHENG Ke-yan; FEI Xiao-fang

2012-01-01

To explore the effects of traditional herbal medicine Ganoderma tsugae(G.tsugae) on immunomodulatory and antitumor activities,the crude polysaccharides ofG.tsugae were purified by filtration,diethylaminoethyl(DEAE)sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography.Two main fractions,protein-containing glycans CSSLP-I and CSSLP-2,were obtained via the gradient elution.The protein content,molecular weight,and monosaccharide composition of the two fractions were analyzed.Furthermore,the influence of the protein-containing glycans from G.tsugae on the activation of human acute monocytic leukemia cell line(THP-1 ) and their antitumor activities to the human hepatocellular liver carcinoma cell(HepG-2) in vitro were evaluated.The results indicate that CSSLP-I and CSSLP-2 could increase the pinocytic activity of THP-1 cells and induce THP-1 cells to produce the cytokines of TNFa and IL-2,significantly.CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell(NepG-2).Moreover,the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the participation of TNFa and 1L-2 or other antitumor factors induced from THP-1 cclls by G.tsugae protein-containing glycan fractions.

The Arabidopsis YUCCA1 Flavin Monooxygenase Functions in the Indole-3-Pyruvic Acid Branch of Auxin Biosynthesis

Czech Academy of Sciences Publication Activity Database

Stepanova, A.N.; Yun, J.; Robles, L.M.; Novák, Ondřej; He, W.; Guo, H.W.; Ljung, K.; Alonso, J.M.

2011-01-01

Roč. 23, č. 11 (2011), s. 3961-3973 ISSN 1040-4651 R&D Projects: GA ČR GA301/08/1649 Keywords : PLANT DEVELOPMENT * GLUCOSINOLATE BIOSYNTHESIS * REPRODUCTIVE DEVELOPMENT * MASS-SPECTROMETRY * ALDEHYDE OXIDASE * THALIANA * GENE * METABOLISM * MUTANTS * PATHWAY Subject RIV: EF - Botanics Impact factor: 8.987, year: 2011

Oxidoreductases on their way to industrial biotransformations

NARCIS (Netherlands)

Martínez, Angel T.; Ruiz-Dueñas, Francisco J.; Camarero, Susana; Serrano, Ana; Linde, Dolores; Lund, Henrik; Vind, Jesper; Tovborg, Morten; Herold-Majumdar, Owik M.; Hofrichter, Martin; Liers, Christiane; Berkel, van Willem J.H.

2017-01-01

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and

Oxidoreductases on their way to industrial biotransformations

NARCIS (Netherlands)

Martínez, Angel T.; Ruiz-Dueñas, Francisco J.; Camarero, Susana; Serrano, Ana; Linde, Dolores; Lund, Henrik; Vind, Jesper; Tovborg, Morten; Herold-Majumdar, Owik M.; Hofrichter, Martin; Liers, Christiane; Ullrich, René; Scheibner, Katrin; Sannia, Giovanni; Piscitelli, Alessandra; Pezzella, Cinzia; Sener, Mehmet E.; Kılıç, Sibel; van Berkel, Willem J.H.; Guallar, Victor; Lucas, Maria Fátima; Zuhse, Ralf; Ludwig, Roland; Hollmann, F.; Fernandez Fueyo, E.; Record, Eric; Faulds, Craig B.; Tortajada, Marta; Winckelmann, Ib; Rasmussen, Jo Anne; Gelo-Pujic, Mirjana; Gutiérrez, Ana; del Río, José C.; Rencoret, Jorge; Alcalde, Miguel

2017-01-01

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases

Nitrogen transformations as inferred from the activities of key enzymes in the Arabian Sea oxygen minimum zone

Digital Repository Service at National Institute of Oceanography (India)

Shailaja, M.S.; Narvekar, P.V.; Alagarsamy, R.; Naqvi, S.W.A.

within the sigma theta range 26.6-26.8, which corresponds to the Persian Gulf Watermass (PGW). Depth profiles of nitrate reductase (NaR), nitrite reductase (NiR) and ammonia monooxygenase (AMO) activities were compared with those of O2, NO3(super...

Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

International Nuclear Information System (INIS)

Roccatano, Danilo

2015-01-01

The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure–dynamics–function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions. (topical review)

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

DEFF Research Database (Denmark)

Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate......-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin...... ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together...

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO).

Science.gov (United States)

Usselman, Robert J; Fielding, Alistair J; Frerman, Frank E; Watmough, Nicholas J; Eaton, Gareth R; Eaton, Sandra S

2008-01-08

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.

Kynurenine 3-monooxygenase is implicated in antidepressants-responsive depressive-like behaviors and monoaminergic dysfunctions.

Science.gov (United States)

Tashiro, Tomoyuki; Murakami, Yuki; Mouri, Akihiro; Imamura, Yukio; Nabeshima, Toshitaka; Yamamoto, Yasuko; Saito, Kuniaki

2017-01-15

l-Tryptophan (TRP) is metabolized via serotonin and kynurenine pathways (KP). Several studies have demonstrated that abnormality of both pathways is involved in the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the KP, has been suggested to play major roles in physiological and pathological events mediated by bioactive kynurenine metabolites. In this study, we investigated the role of KMO in the emotional and cognitive functions by using KMO knockout (KO) mice. We measured contents of TRP and monoamines and their metabolites in the serum and hippocampus of KMO KO mice. Further, we investigated whether antidepressants improved the depressive-like behaviors in KMO KO mice. KMO KO mice showed depressive-like behaviors such as decreased sucrose preference and increased immobility in the forced swimming test and high anxiety by decreased time spent in the center area of open field. But, there was no difference in spontaneous alternation in Y-maze test, counts of rearing or locomotor activity. Higher contents of TRP metabolites such as kynurenine (KYN), kynurenic acid (KA), anthranilic acid (AA), and 3-hydroxykynurenine (3-HK) in the serum and hippocampus and decreased serotonin turnover and higher content of normetanephrine (NM) in the hippocampus were observed in the KMO KO mice. Although both antidepressant attenuated increase of immobility, sertraline but not imipramine improved decrease of sucrose preference in the KMO KO mice. These findings suggested that KMO KO mice show antidepressants-responsive depressive-like behaviors and monoaminergic dysfunctions via abnormality of kynurenine metabolism with good validities as MDD model. Copyright © 2016 Elsevier B.V. All rights reserved.

A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

Science.gov (United States)

Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

2015-09-01

Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of bottlenecks for P450 biotransformation processes

DEFF Research Database (Denmark)

Andersson, Marie Therese; Törnvall, Ulrika; Tufvesson, Pär

Cytochrome P450 monooxygenases (P450 or CYP) is a group of heme-containing enzymes hydroxylating non-activated hydrocarbons in a stereospecific manner, something that is hard to achieve via classical chemistry. The importance of these reactions can be stressed by the hydroxylation of steroids, bu...... biotransformation process identifying the limiting parameters and defining relevant targets....

Impact of Mutations on the Midpoint Potential of the [4Fe-4S]+1,+2 Cluster and on Catalytic Activity in Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)†

Science.gov (United States)

Usselman, Robert J.; Fielding, Alistair J.; Frerman, Frank E.; Watmough, Nicholas J.; Eaton, Gareth R.; Eaton, Sandra S.

2011-01-01

Electron transfer flavoprotein - ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen bonding distance of the Sγ of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the mid-point potentials of the iron-sulfur cluster from +37 mV for wild type to −60 mV for Y501F and T525A and to −128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials. PMID:18069858

Pipecolic Acid Orchestrates Plant Systemic Acquired Resistance and Defense Priming via Salicylic Acid-Dependent and -Independent Pathways.

Science.gov (United States)

Bernsdorff, Friederike; Döring, Anne-Christin; Gruner, Katrin; Schuck, Stefan; Bräutigam, Andrea; Zeier, Jürgen

2016-01-01

We investigated the relationships of the two immune-regulatory plant metabolites, salicylic acid (SA) and pipecolic acid (Pip), in the establishment of plant systemic acquired resistance (SAR), SAR-associated defense priming, and basal immunity. Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and Pip, we show that SA and Pip act both independently from each other and synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae. Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by preactivating multiple stages of defense signaling and that SA accumulation upon SAR activation leads to the downregulation of photosynthesis and attenuated jasmonate responses systemically within the plant. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. During SAR, Pip orchestrates SA-dependent and SA-independent priming of pathogen responses in a FLAVIN-DEPENDENT-MONOOXYGENASE1 (FMO1)-dependent manner. We conclude that a Pip/FMO1 signaling module acts as an indispensable switch for the activation of SAR and associated defense priming events and that SA amplifies Pip-triggered responses to different degrees in the distal tissue of SAR-activated plants. © 2016 American Society of Plant Biologists. All rights reserved.

First molecular modeling report on novel arylpyrimidine kynurenine monooxygenase inhibitors through multi-QSAR analysis against Huntington's disease: A proposal to chemists!

Science.gov (United States)

Amin, Sk Abdul; Adhikari, Nilanjan; Jha, Tarun; Gayen, Shovanlal

2016-12-01

Huntington's disease (HD) is caused by mutation of huntingtin protein (mHtt) leading to neuronal cell death. The mHtt induced toxicity can be rescued by inhibiting the kynurenine monooxygenase (KMO) enzyme. Therefore, KMO is a promising drug target to address the neurodegenerative disorders such as Huntington's diseases. Fiftysix arylpyrimidine KMO inhibitors are structurally explored through regression and classification based multi-QSAR modeling, pharmacophore mapping and molecular docking approaches. Moreover, ten new compounds are proposed and validated through the modeling that may be effective in accelerating Huntington's disease drug discovery efforts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism.

Science.gov (United States)

Furuya, Toshiki; Nakao, Tomomi; Kino, Kuniki

2015-10-01

Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The development and testing of a modular containment system under plutonium active conditions

International Nuclear Information System (INIS)

Sanders, M.J.; Pengelly, M.G.A.

1984-05-01

A Modular Containment System has been designed, constructed and tested under plutonium active conditions at AEE Winfrith. The unit consists of a portable self-contained pressurised suit area, complete with shower entry tunnel and ventilation plant which can be assembled to enclose active plant to enable active operations to be carried out safely by operators dressed in standard pressurised suits. A fundamental feature of the system is the use of strippable coatings which are used to treat the interior surfaces prior to active operations to prevent permanent contamination of the structure. Details of construction are given together with results of trials. Whilst this report describes work with plutonium, the system has clear applications wherever temporary containment of radioactive or toxic materials is needed. (U.K.)

CYP79 P450 monooxygenases in gymnosperms: CYP79A118 is associated with the formation of taxiphyllin in Taxus baccata.

Science.gov (United States)

Luck, Katrin; Jia, Qidong; Huber, Meret; Handrick, Vinzenz; Wong, Gane Ka-Shu; Nelson, David R; Chen, Feng; Gershenzon, Jonathan; Köllner, Tobias G

2017-09-01

Conifers contain P450 enzymes from the CYP79 family that are involved in cyanogenic glycoside biosynthesis. Cyanogenic glycosides are secondary plant compounds that are widespread in the plant kingdom. Their biosynthesis starts with the conversion of aromatic or aliphatic amino acids into their respective aldoximes, catalysed by N-hydroxylating cytochrome P450 monooxygenases (CYP) of the CYP79 family. While CYP79s are well known in angiosperms, their occurrence in gymnosperms and other plant divisions containing cyanogenic glycoside-producing plants has not been reported so far. We screened the transcriptomes of 72 conifer species to identify putative CYP79 genes in this plant division. From the seven resulting full-length genes, CYP79A118 from European yew (Taxus baccata) was chosen for further characterization. Recombinant CYP79A118 produced in yeast was able to convert L-tyrosine, L-tryptophan, and L-phenylalanine into p-hydroxyphenylacetaldoxime, indole-3-acetaldoxime, and phenylacetaldoxime, respectively. However, the kinetic parameters of the enzyme and transient expression of CYP79A118 in Nicotiana benthamiana indicate that L-tyrosine is the preferred substrate in vivo. Consistent with these findings, taxiphyllin, which is derived from L-tyrosine, was the only cyanogenic glycoside found in the different organs of T. baccata. Taxiphyllin showed highest accumulation in leaves and twigs, moderate accumulation in roots, and only trace accumulation in seeds and the aril. Quantitative real-time PCR revealed that CYP79A118 was expressed in plant organs rich in taxiphyllin. Our data show that CYP79s represent an ancient family of plant P450s that evolved prior to the separation of gymnosperms and angiosperms. CYP79A118 from T. baccata has typical CYP79 properties and its substrate specificity and spatial gene expression pattern suggest that the enzyme contributes to the formation of taxiphyllin in this plant species.

Physical properties of highly active liquor containing molybdate solids

International Nuclear Information System (INIS)

Dunnett, B.; Ward, T.; Roberts, R.; Cheeseright, J.

2016-01-01

The reprocessing of irradiated nuclear fuel at Sellafield produces a nitric acid based Highly Active Liquor (HAL) waste. The liquor, containing fission products and process additives, is concentrated in an evaporator in order to reduce the volume and is then stored in Highly Active Storage Tanks (HASTs) prior to vitrification. Caesium phosphomolybdate (CPM) is precipitated during the evaporation process and can convert to zirconium molybdate (ZM) during storage. During Post Operational Clean Out (POCO) of the HASTs, it is expected that their highly active content will be reduced by repeated cycles of washing using nitric acid and other reagents. Initial washings are likely to have a chemical composition comparable to concentrated HAL, becoming more dilute during the wash-out process. It is expected that the wash-out process will also recover significant quantities of molybdate solids (ZM, CPM or a mixture) from the HASTs. In order to determine the processing challenges from such washings during POCO, the physical properties of varying concentrations of non-active HAL simulants containing molybdate solids have recently been measured by the UK's National Nuclear Laboratory. The following measurements are presented and discussed: Particle size distribution; Density; Settling behaviour of solids; Voidage of settled sediment beds; Viscosity; Yield stress; And influence of ZM morphology on physical properties. (authors)

Physical properties of highly active liquor containing molybdate solids

Energy Technology Data Exchange (ETDEWEB)

Dunnett, B.; Ward, T.; Roberts, R. [National Nuclear Laboratory, Sellafield, Seascale, Cumbria, CA20 1PG (United Kingdom); Cheeseright, J. [Sellafield Ltd, Sellafield, Seascale, Cumbria, CA20 1PG (United Kingdom)

2016-07-01

The reprocessing of irradiated nuclear fuel at Sellafield produces a nitric acid based Highly Active Liquor (HAL) waste. The liquor, containing fission products and process additives, is concentrated in an evaporator in order to reduce the volume and is then stored in Highly Active Storage Tanks (HASTs) prior to vitrification. Caesium phosphomolybdate (CPM) is precipitated during the evaporation process and can convert to zirconium molybdate (ZM) during storage. During Post Operational Clean Out (POCO) of the HASTs, it is expected that their highly active content will be reduced by repeated cycles of washing using nitric acid and other reagents. Initial washings are likely to have a chemical composition comparable to concentrated HAL, becoming more dilute during the wash-out process. It is expected that the wash-out process will also recover significant quantities of molybdate solids (ZM, CPM or a mixture) from the HASTs. In order to determine the processing challenges from such washings during POCO, the physical properties of varying concentrations of non-active HAL simulants containing molybdate solids have recently been measured by the UK's National Nuclear Laboratory. The following measurements are presented and discussed: Particle size distribution; Density; Settling behaviour of solids; Voidage of settled sediment beds; Viscosity; Yield stress; And influence of ZM morphology on physical properties. (authors)

What is the contribution of human FMO3 in the N-oxygenation of selected therapeutic drugs and drugs of abuse?

Science.gov (United States)

Wagmann, Lea; Meyer, Markus R; Maurer, Hans H

2016-09-06

Little is known about the role of flavin-containing monooxygenases (FMOs) in the metabolism of xenobiotics. FMO3 is the isoform in adult human liver with the highest impact on drug metabolism. The aim of the presented study was to elucidate the contribution of human FMO3 to the N-oxygenation of selected therapeutic drugs and drugs of abuse (DOAs). Its contribution to the in vivo hepatic net clearance of the N-oxygenation products was calculated by application of an extended relative activity factor (RAF) approach to differentiate from contribution of cytochrome P450 (CYP) isoforms. FMO3 and CYP substrates were identified using pooled human liver microsomes after heat inactivation and chemical inhibition, or single enzyme incubations. Kinetic parameters were subsequently determined using recombinant human enzymes and mass spectrometric analysis via authentic reference standards or simple peak areas of the products divided by those of the internal standard. FMO3 was identified as enzyme mainly responsible for the formation of N,N-diallyltryptamine N-oxide and methamphetamine hydroxylamine (>80% contribution for both). A contribution of 50 and 30% was calculated for the formation of N,N-dimethyltryptamine N-oxide and methoxypiperamide N-oxide, respectively. However, FMO3 contributed with less than 5% to the formation of 3-bromomethcathinone hydroxylamine, amitriptyline N-oxide, and clozapine N-oxide. There was no significant difference in the contributions when using calibrations with reference metabolite standards or peak area ratio calculations. The successful application of a modified RAF approach including FMO3 proved the importance of FMO3 in the N-oxygenation of DOAs in human metabolism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A fraction enriched in rat hippocampal mossy fibre synaptosomes contains trophic activities.

Science.gov (United States)

Taupin, P; Roisin, M P; Ben-Ari, Y; Barbin, G

1994-06-27

Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.

Trimethylamine N-Oxide: The Good, the Bad and the Unknown

Directory of Open Access Journals (Sweden)

Manuel T. Velasquez

2016-11-01

Full Text Available Trimethylamine N-oxide (TMAO is a small colorless amine oxide generated from choline, betaine, and carnitine by gut microbial metabolism. It accumulates in the tissue of marine animals in high concentrations and protects against the protein-destabilizing effects of urea. Plasma level of TMAO is determined by a number of factors including diet, gut microbial flora and liver flavin monooxygenase activity. In humans, a positive correlation between elevated plasma levels of TMAO and an increased risk for major adverse cardiovascular events and death is reported. The atherogenic effect of TMAO is attributed to alterations in cholesterol and bile acid metabolism, activation of inflammatory pathways and promotion foam cell formation. TMAO levels increase with decreasing levels of kidney function and is associated with mortality in patients with chronic kidney disease. A number of therapeutic strategies are being explored to reduce TMAO levels, including use of oral broad spectrum antibiotics, promoting the growth of bacteria that utilize TMAO as substrate and the development of target-specific molecules with varying level of success. Despite the accumulating evidence, it is questioned whether TMAO is the mediator of a bystander in the disease process. Thus, it is important to undertake studies examining the cellular signaling in physiology and pathological states in order to establish the role of TMAO in health and disease in humans.

Characterization of alternate reductant binding and electron transfer in the dopamine β-monooxygenase reaction

International Nuclear Information System (INIS)

Stewart, L.C.; Klinman, J.P.

1987-01-01

The steady-state limiting kinetic parameters V/sub max/, V/K/sub DA/, and V/K/sub O 2 /, together with deuterium isotope effects on these parameters, have been determined for the dopamine β-monooxygenase (DβM) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascrobate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of V/sub max/ and V/K/sub DA/, and the authors conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/K/sub O 2 / have been measured with [2,2- 2 H 2 ]dopamine as substrate both in the presence and in the absence of added ascorbate. This key result unambiguously rules out an entry of electrons to enzyme forms leading from the enzyme-dopamine complex to enzyme-bound product and, hence, reaction mechanisms involving a reductive activation of the putative Cu(II)-OOH prior to substrate hydroxylation

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of “Desulfomonile tiedjei”

OpenAIRE

DeWeerd, Kim A.; Suflita, Joseph M.

1990-01-01

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, “Desulfomonile tiedjei.” We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c3, or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, C...

ADMET Polymerization Activities of Electrochemically Reduced W-Based Active Species for Ge- and Sn-Containing Dienes

Science.gov (United States)

Imamoglu, Yavuz; Aydogdu, Cemil; Karabulut, Solmaz; Düz, Bülent

In the last 20 years metal atom-containing polymers have become important classes of polymers [1]. Properties like high thermic stability, electric, and photo conductometry make them very interesting for producing films, fibers, and coating [2]. Many of these compounds can be synthesized by conventional methods [3]. For producing metal-containing polymers anionic, cationic, and radicalic polymerizations were used [4-6]. Metal-containing polymers were also synthesized via acyclic diene metathesis (ADMET) polymerization that is facilitated by Schrock’s molybdenum alkylidene, or Grubbs’ ruthenium carbene catalyst [7-9]. In 1979, Gilet and coworkers succeeded in synthesizing metathetically active species from electrochemical reduction of WCl6 and MoCl5 [10,11]. In the light of these works, we have showed that electrochemically generated tungsten-based active species (WCl6-e--Al-CH2Cl2) catalyzes various metathesis-related reactions [12-16].

The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3.

Science.gov (United States)

Nikodinovic-Runic, Jasmina; Coulombel, Lydie; Francuski, Djordje; Sharma, Narain D; Boyd, Derek R; Ferrall, Rory Moore O; O'Connor, Kevin E

2013-06-01

Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.

Development of a Series of Kynurenine 3-Monooxygenase Inhibitors Leading to a Clinical Candidate for the Treatment of Acute Pancreatitis.

Science.gov (United States)

Walker, Ann L; Ancellin, Nicolas; Beaufils, Benjamin; Bergeal, Marylise; Binnie, Margaret; Bouillot, Anne; Clapham, David; Denis, Alexis; Haslam, Carl P; Holmes, Duncan S; Hutchinson, Jonathan P; Liddle, John; McBride, Andrew; Mirguet, Olivier; Mowat, Christopher G; Rowland, Paul; Tiberghien, Nathalie; Trottet, Lionel; Uings, Iain; Webster, Scott P; Zheng, Xiaozhong; Mole, Damian J

2017-04-27

Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.

Do microbial exudates control EH electrode measurements?

Science.gov (United States)

Markelova, E.; Parsons, C. T.; Smeaton, C. M.; Van Cappellen, P.

2017-12-01

Redox electrodes are widely used as simple, inexpensive monitoring devices to rapidly measure redox potentials (EH) of waterlogged soils, sediments, and aquifers. While a variety of physicochemical and biogeochemical factors have been involved to explain measured EH values, the role of microorganisms remains comparatively understudied and uncertain. Besides catalyzing many inorganic redox reactions (e.g., nitrate reduction), microorganisms produce a variety of redox-active organic compounds (e.g., NAD+/NADH, GSSG/2GSH, FAD/FADH2), which can be released into the surrounding environment via active secretion, passive diffusion, or cell lysis. To isolate different microbial effects on EH measurements, we performed batch experiments using S. oneidensis MR-I as a model heterotrophic microorganism and flavins as example microbial exudates [1]. We monitored EH and pH along with flavin production (fluorescence measurements) during dissimilatory nitrate reduction to ammonium (DNRA). Dissolved flavins increased to 0.2 mM (riboflavin equivalent) under anoxic conditions during complete consumption of 1 mM nitrate by DNRA at pH 7.4 and 30 °C over 80 hours. The observed redox cascade from +255 to -250 mV did not follow the EH predicted for the reduction of NO3- to NO2- and NO2- to NH4+ by the Nernst equation. However, a set of separate abiotic experiments on the photoreduction of synthetic flavins (LMC, RF, FMN, and FAD, Sigma Aldrich) under the same conditions indicated that measured EH values are buffered at +270 ± 20 mV and -230 ± 50 mV when oxidized and reduced flavin species dominate, respectively. Moreover, based on the temporal changes in EH, we speculate that NO3- reduction by S. oneidensis consumes reduced flavins (i.e., NO3- accepts electrons from reduced flavins) and generates oxidized flavins, thus buffering EH at +255 mV. By contrast, NO2- reduction to NH4+ is independent of flavin speciation, which leads to the accumulation of reduced flavins in the solution and

Conclusive report on the activities of the Containment Expert Group 1975-87

International Nuclear Information System (INIS)

Balz, W.; Dufresne, J.

1988-01-01

The report summarizes the results of the activities of the CONT Expert Group, a subgroup of the CEC Fast Reactor Safety Working Group (SWG). The CONT group's work has over the last 12 years (1975-87) covered a wide spectrum of problems related to the behaviour of the primary containment of a sodium-cooled fast reactor following an accident releasing a large amount of mechanical energy. In particular the CONT group followed closely the code development and validation (COVA) programme carried out at the JRC Ispra. This activity also included an assessment of related material programmes. From a comparison of containment codes and a sensitivity analysis, it was concluded that the codes developed in the EC Member States and at the JRC Ispra allow to treat the consequences of a hypothetical core-disruptive accident on primary containment with adequate accuracy. Consequently the the group considered its mandate fulfilled and terminated its activities

Blue and ultraviolet-B light photoreceptors in parsley cells

International Nuclear Information System (INIS)

Ensminger, P.A.; Schaefer, E.

1992-01-01

The authors studied UV-B photoreception in parsley cell cultures with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photo-reception. Cells irradiated with UV-B light (0.5-15 min) at 2 o C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25 o C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. (author)

Green autofluorescence, a double edged monitoring tool for bacterial growth and activity in micro-plates

Science.gov (United States)

Mihalcescu, Irina; Van-Melle Gateau, Mathilde; Chelli, Bernard; Pinel, Corinne; Ravanat, Jean-Luc

2015-12-01

The intrinsic green autofluorescence of an Escherichia coli culture has long been overlooked and empirically corrected in green fluorescent protein (GFP) reporter experiments. We show here, by using complementary methods of fluorescence analysis and HPLC, that this autofluorescence, principally arise from the secreted flavins in the external media. The cells secrete roughly 10 times more than what they keep inside. We show next that the secreted flavin fluorescence can be used as a complementary method in measuring the cell concentration particularly when the classical method, based on optical density measure, starts to fail. We also demonstrate that the same external flavins limit the dynamical range of GFP quantification and can lead to a false interpretation of lower global dynamic range of expression than what really happens. In the end we evaluate different autofluorescence correction methods to extract the real GFP signal.

Targeted deletion of kynurenine 3-monooxygenase in mice: a new tool for studying kynurenine pathway metabolism in periphery and brain.

Science.gov (United States)

Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V; Notarangelo, Francesca M; Thomas, Marian A R; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J

2013-12-20

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo(-/-) mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo(-/-) mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo(-/-) mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD(+), did not differ between Kmo(-/-) and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo(-/-) mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo(-/-) mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.

Diversity of the active methanotrophic community in acidic peatlands as assessed by mRNA and SIP-PLFA analyses.

Science.gov (United States)

Chen, Yin; Dumont, Marc G; McNamara, Niall P; Chamberlain, Paul M; Bodrossy, Levente; Stralis-Pavese, Nancy; Murrell, J Colin

2008-02-01

The active methanotroph community was investigated for the first time in heather (Calluna)-covered moorlands and Sphagnum/Eriophorum-covered UK peatlands. Direct extraction of mRNA from these soils facilitated detection of expression of methane monooxygenase genes, which revealed that particulate methane monooxygenase and not soluble methane monooxygenase was probably responsible for CH(4) oxidation in situ, because only pmoA transcripts (encoding a subunit of particulate methane monooxygenase) were readily detectable. Differences in methanotroph community structures were observed between the Calluna-covered moorland and Sphagnum/Eriophorum-covered gully habitats. As with many other Sphagnum-covered peatlands, the Sphagnum/Eriophorum-covered gullies were dominated by Methylocystis. Methylocella and Methylocapsa-related species were also present. Methylobacter-related species were found as demonstrated by the use of a pmoA-based diagnostic microarray. In Calluna-covered moorlands, in addition to Methylocella and Methylocystis, a unique group of peat-associated type I methanotrophs (Gammaproteobacteria) and a group of uncultivated type II methanotrophs (Alphaproteobacteria) were also found. The pmoA sequences of the latter were only distantly related to Methylocapsa and also to the RA-14 group of methanotrophs, which are believed to be involved in oxidation of atmospheric concentrations of CH(4). Soil samples were also labelled with (13)CH(4), and subsequent analysis of the (13)C-labelled phospholipid fatty acids (PLFAs) showed that 16:1 omega 7, 18:1 omega 7 and 18:1 omega 9 were the major labelled PLFAs. The presence of (13)C-labelled 18:1 omega 9, which was not a major PLFA of any extant methanotrophs, indicated the presence of novel methanotrophs in this peatland.

Independent Recruitment of a Flavin-Dependent Monooxygenase for Safe Accumulation of Sequestered Pyrrolizidine Alkaloids in Grasshoppers and Moths

OpenAIRE

Wang, Linzhu; Beuerle, Till; Timbilla, James; Ober, Dietrich

2012-01-01

Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide,...

Yucca mountain container fabrication, closure and non-destructive evaluation development activities

International Nuclear Information System (INIS)

Russell, E.W.; Nelson, T.A.

1990-01-01

Container fabrication, closure, and non-destructive evaluation process development activities are described. The design parameters for a tuff environment are: no significant hydrostatic or lithostatic loading of the container; very small water flux; benign water, an oxidizing, dilute sodium bicarbonate solution of neutral pH; temperatures reaching 250 C over the first 50 to 100 years, then falling to about 97 C over the remainder of the 300-year container period. The materials under consideration are three austenitic alloys: AISI 304L, AISI 316L, and alloy 825; as well as three copper alloys: CDA 102, CDA 613, and CDA 715. Targets are controlled, uniform microstructures for the base metal, the weld and the heat affected zones of the weld; controlled microchemistry; low residual stresses; small welds and heat-affected zones; and reliable methods of flaw detection by surface and volumetric activities. The recommended procedures and processes for fabricating, closing and evaluating each of the candidate materials are reviewed

A container for containing and protecting a radioactive substance

International Nuclear Information System (INIS)

1974-01-01

The invention relates to a container adapted to contain and protect a radio-active substance. That container comprises a heat sensitive device for automatically (and, preferably, sealingly) enclosing and protecting the radio-active substance, should room temperature reach a predetermined level. Thus, the radio-active substance cannot escape in case of fire. Preferably, a bolt is also provided, capable of being actuated at a temperature slightly above the temperature actuating the protective device so as to maintain the radioactive substance protected. This can be applied to containers containing a radio-active substance such as polonium 210 [fr

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

Science.gov (United States)

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

Science.gov (United States)

Jacobs, K R; Guillemin, G J; Lovejoy, D B

2018-02-01

Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

77 FR 23490 - Agency Information Collection Activities: Country of Origin Marking Requirements for Containers...

Science.gov (United States)

2012-04-19

... Activities: Country of Origin Marking Requirements for Containers or Holders AGENCY: U.S. Customs and Border... of Origin Marking Requirements for Containers or Holders. This is a proposed extension of an... Requirements for Containers or Holders. [[Page 23491

Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids

Science.gov (United States)

Summers, Ryan M.; Louie, Tai Man; Yu, Chi-Li; Gakhar, Lokesh; Louie, Kailin C.

2012-01-01

The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N1- and N3-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His6 fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His6 plus His6-NdmD catalyzed N1-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His6 plus His6-NdmD catalyzed N3-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N7-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste. PMID:22328667

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

Science.gov (United States)

Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

2013-11-01

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

Description of a Riboflavin Biosynthetic Gene Variant Prevalent in the Phylum Proteobacteria

Science.gov (United States)

Brutinel, Evan D.; Dean, Antony M.

2013-01-01

Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide, which are cofactors essential for a host of intracellular redox reactions. Microorganisms synthesize flavins de novo to fulfill nutritional requirements, but it is becoming increasingly clear that flavins play a wider role in cellular physiology than was previously appreciated. Flavins mediate diverse processes beyond the cytoplasmic membrane, including iron acquisition, extracellular respiration, and interspecies interactions. While investigating the regulation of flavin electron shuttle biosynthesis in the Gram-negative gammaproteobacterium Shewanella oneidensis, we discovered that a riboflavin biosynthetic gene (ribBA) annotated as encoding a bifunctional 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase/GTP cyclohydrolase II does not possess both functions. The novel gene, renamed ribBX here, encodes an amino-terminal DHBP synthase domain. The carboxy-terminal end of RibBX not only lacks GTP cyclohydrolase II activity but also has evolved a different function altogether in S. oneidensis, regulating the activity of the DHBP synthase domain. Phylogenetic analysis revealed that the misannotation of ribBX as ribBA is rampant throughout the phylum Proteobacteria (40% of 2,173 annotated ribBA genes) and that ribBX emerged early in the evolution of this group of microorganisms. We examined the functionality of representative ribBX genes from Beta-, Gamma-, and Epsilonproteobacteria and found that, consistent with sequence-based predictions, the encoded GTP cyclohydrolase II domains lack catalytic activity. The persistence of ribBX in the genomes of so many phylogenetically divergent bacterial species lends weight to the argument that ribBX has evolved a function which lends a selective advantage to the host. PMID:24097946

77 FR 6135 - Agency Information Collection Activities: Cargo Manifest/Declaration, Stow Plan, Container Status...

Science.gov (United States)

2012-02-07

... Activities: Cargo Manifest/ Declaration, Stow Plan, Container Status Messages and Importer Security Filing... the Paperwork Reduction Act: Cargo Manifest/Declaration, Stow Plan, Container Status Messages and.../Declaration, Stow Plan, Container Status Messages and Importer Security Filing. OMB Number: 1651-0001. Form...

Processing method and processing device for liquid waste containing surface active agent and radioactive material

International Nuclear Information System (INIS)

Nishi, Takashi; Matsuda, Masami; Baba, Tsutomu; Yoshikawa, Ryozo; Yukita, Atsushi.

1998-01-01

Washing liquid wastes containing surface active agents and radioactive materials are sent to a deaerating vessel. Ozone is blown into the deaerating vessel. The washing liquid wastes dissolved with ozone are introduced to a UV ray irradiation vessel. UV rays are irradiated to the washing liquid wastes, and hydroxy radicals generated by photodecomposition of dissolved ozone oxidatively decompose surface active agents contained in the washing liquid wastes. The washing liquid wastes discharged from the UV ray irradiation vessel are sent to an activated carbon mixing vessel and mixed with powdery activated carbon. The surface active agents not decomposed in the UV ray irradiation vessel are adsorbed to the activated carbon. Then, the activated carbon and washing liquid wastes are separated by an activated carbon separating/drying device. Radioactive materials (iron oxide and the like) contained in the washing liquid wastes are mostly granular, and they are separated and removed from the washing liquid wastes in the activated carbon separating/drying device. (I.N.)

Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

Science.gov (United States)

Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

2017-08-18

Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of resource activities upon repository siting and waste containment with reference to bedded salt

International Nuclear Information System (INIS)

Ashby, J.; Rowe, J.

1980-02-01

The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases

Methylocapsa aurea sp. nov., a facultative methanotroph possessing a particulate methane monooxygenase, and emended description of the genus Methylocapsa.

Science.gov (United States)

Dunfield, Peter F; Belova, Svetlana E; Vorob'ev, Alexey V; Cornish, Sabrina L; Dedysh, Svetlana N

2010-11-01

An aerobic, methanotrophic bacterium, designated KYG(T), was isolated from a forest soil in Germany. Cells of strain KYG(T) were Gram-negative, non-motile, slightly curved rods that multiplied by binary fission and produced yellow colonies. The cells contained intracellular granules of poly-β-hydroxybutyrate at each cell pole, a particulate methane monooxygenase (pMMO) and stacks of intracytoplasmic membranes (ICMs) packed in parallel along one side of the cell envelope. Strain KYG(T) grew at pH 5.2-7.2 and 2-33 °C and could fix atmospheric nitrogen under reduced oxygen tension. The major cellular fatty acid was C(18 : 1)ω7c (81.5 %) and the DNA G+C content was 61.4 mol%. Strain KYG(T) belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the obligate methanotroph Methylocapsa acidiphila B2(T) (98.1 % 16S rRNA gene sequence similarity and 84.7 % pmoA sequence similarity). Unlike Methylocapsa acidiphila B2(T), which grows only on methane and methanol, strain KYG(T) was able to grow facultatively on acetate. Facultative acetate utilization is a characteristic of the methanotrophs of the genus Methylocella, but the genus Methylocella does not produce pMMO or ICMs. Strain KYG(T) differed from Methylocapsa acidiphila B2(T) on the basis of substrate utilization pattern, pigmentation, pH range, cell ultrastructure and efficiency of dinitrogen fixation. Therefore, we propose a novel species, Methylocapsa aurea sp. nov., to accommodate this bacterium. The type strain is KYG(T) (=DSM 22158(T) =VKM B-2544(T)).

Development of active biofilms of quinoa (Chenopodium quinoa W.) starch containing gold nanoparticles and evaluation of antimicrobial activity.

Science.gov (United States)

Pagno, Carlos H; Costa, Tania M H; de Menezes, Eliana W; Benvenutti, Edilson V; Hertz, Plinho F; Matte, Carla R; Tosati, Juliano V; Monteiro, Alcilene R; Rios, Alessandro O; Flôres, Simone H

2015-04-15

Active biofilms of quinoa (Chenopodium quinoa, W.) starch were prepared by incorporating gold nanoparticles stabilised by an ionic silsesquioxane that contains the 1,4-diazoniabicyclo[2.2.2]octane chloride group. The biofilms were characterised and their antimicrobial activity was evaluated against Escherichiacoli and Staphylococcusaureus. The presence of gold nanoparticles produces an improvement in the mechanical, optical and morphological properties, maintaining the thermal and barrier properties unchanged when compared to the standard biofilm. The active biofilms exhibited strong antibacterial activity against food-borne pathogens with inhibition percentages of 99% against E. coli and 98% against S. aureus. These quinoa starch biofilms containing gold nanoparticles are very promising to be used as active food packaging for the maintenance of food safety and extension of the shelf life of packaged foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Containment

International Nuclear Information System (INIS)

Anon.

1987-01-01

The primary mission of the Containment Group is to ensure that underground nuclear tests are satisfactorily contained. The main goal is the development of sound technical bases for containment-related methodology. Major areas of activity include siting, geologic description, emplacement hole stemming, and phenomenological predictions. Performance results of sanded gypsum concrete plugs on the Jefferson, Panamint, Cornucopia, Labquark, and Bodie events are given. Activities are also described in the following areas: computational capabilities site description, predictive modeling, and cavity-pressure measurement. Containment publications are listed. 8 references

'Becquerel screening' device to automatically measure activity level of contaminated soil in flexible containers

International Nuclear Information System (INIS)

Okada, Hisashi; Yamaguchi, Yoshihisa; Yamamoto, Shuji

2014-01-01

Contaminated soil and incineration ash collected during offsite decontamination work following the accident at the Fukushima Daiichi Nuclear Power Station of Tokyo Electric Power Co., Inc. is being stored in flexible containers. These containers are managed taking their activity level into account in accordance with the Decontamination Guidelines issued by the Ministry of the Environment. Toshiba has developed the 'Becquerel Screening' device that can automatically measure the activity level of the contents of each flexible container simply by placing the container on a palette, without the need to take samples for analysis from the container. The Becquerel Screening device is expected to contribute not only to improved operational efficiency but also to reduced exposure of operators to radiation, because it eliminates the need for direct contact with contaminated soil and ash. (author)

Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains.

Science.gov (United States)

Sharp, Jonathan O; Wood, Thomas K; Alvarez-Cohen, Lisa

2005-03-05

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure. 2005 Wiley Periodicals, Inc.

Catalytic activity of zeolite-containing catalysts in cumene cracking

Energy Technology Data Exchange (ETDEWEB)

Koval' chuk, L V; Takhtarova, G N; Topchieva, K V [Moskovskij Gosudarstvennyj Univ. (USSR). Kafedra Fizicheskoj Khimii

1977-01-01

The catalytic properties are studied of decationized forms (Ca and La) of zeolite-containing catalysts in relation to the nature of the cation and the degree of exchange in the cumene cracking reaction. It has been established that the increase in the activity of Ca-decationized catalysts occurs at a degree of exchange from 22 to 40% and at a ratio of the cation and decationized areas from 0.4 to 1. For La-decationized catalysts the activity increases at a degree of exchange up to 60% and at a ratio between the cation and decationized areas exceeding 1.

77 FR 6815 - Agency Information Collection Activities: Country of Origin Marking Requirements for Containers...

Science.gov (United States)

2012-02-09

... Activities: Country of Origin Marking Requirements for Containers or Holders AGENCY: U.S. Customs and Border... information collection requirement concerning Country of Origin Marking Requirements for Containers or Holders...: Title: Country of Origin Marking Requirements for Containers or Holders. OMB Number: 1651-0057. Form...

Prognostic significance of kynurenine 3-monooxygenase and effects on proliferation, migration, and invasion of human hepatocellular carcinoma.

Science.gov (United States)

Jin, Haojie; Zhang, Yurong; You, Haiyan; Tao, Xuemei; Wang, Cun; Jin, Guangzhi; Wang, Ning; Ruan, Haoyu; Gu, Dishui; Huo, Xisong; Cong, Wenming; Qin, Wenxin

2015-06-23

Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the kynurenine pathway of tryptophan degradation and plays a critical role in Huntington's and Alzheimer's diseases. This study aimed to examine the expression of KMO in human hepatocellular carcinoma (HCC) and investigate the relationship between its expression and prognosis of HCC patients. We first analyzed KMO expression in 120 paired HCC samples (HCC tissues vs matched adjacent non-cancerous liver tissues), and 205 clinical HCC specimens using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis showed that KMO expression was significantly higher in HCC tissues than that in normal liver tissues (all p KMO was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR) (both pKMO positively regulated proliferation, migration, and invasion of HCC cells. These results suggest that KMO exhibits tumor-promoting effects towards HCC and it may serve as a novel prognostic marker in HCC.

Differential Reactivity between Two Copper Sites in Peptidylglycine r-Hydroxylating Monooxygenase

Energy Technology Data Exchange (ETDEWEB)

E Chufan; S Prigge; X Siebert; B Eipper; R Mains; L Amzel

2011-12-31

Peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the C{alpha} of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu{sub M} and Cu{sub H}) separated by {approx}11 {angstrom}. In the resting state of the enzyme, Cu{sub M} is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu{sub H} has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu{sub M} is identified as the metal involved in catalysis, while Cu{sub H} is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO{sub 2}{sup -}), azide (N{sub 3}{sup -}), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO{sub 2}, shows that nitrite anion coordinates Cu{sub M} in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu{sub H}, despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu{sub M} but not Cu{sub H} in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN{sub 3} and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu{sub H} is also observed in the reduced form of the enzyme: CO binds Cu{sub M} but not Cu{sub H} in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5

Biotransformation and induction: implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish

International Nuclear Information System (INIS)

Kleinow, K.M.; Melancon, M.J.; Lech, J.J.

1987-01-01

Biotransformation of xenobiotics in fish occurs by many of the same reactions as in mammals. These reactions have been shown to affect the bioaccumulation, persistence, residue dynamics, and toxicity of select chemicals in fish. P-450-dependent monooxygenase activity of fish can be induced by polycyclic aromatic hydrocarbons, but phenobarbital-type agents induce poorly, if at all. Fish monooxygenase activity exhibits ideal temperature compensation and sex-related variation. Induction of monooxygenase activity by polycyclic aromatic hydrocarbons can result in qualitative as well as quantitative changes in the metabolic profile of a chemical. Induction can also alter toxicity. In addition, multiple P-450 isozymes have been described for several fish species. The biotransformation productions of certain chemicals have been related to specific P-450 isozymes, and the formation of these products can be influenced by induction. Exposure of fish to low levels of certain environmental contaminants has resulted in induction of specific monooxygenase activities and monitoring of such activities has been suggested as a means of identifying areas of pollutant exposure in the wild

sEMG during Whole-Body Vibration Contains Motion Artifacts and Reflex Activity

Directory of Open Access Journals (Sweden)

Karin Lienhard

2015-01-01

Full Text Available The purpose of this study was to determine whether the excessive spikes observed in the surface electromyography (sEMG spectrum recorded during whole-body vibration (WBV exercises contain motion artifacts and/or reflex activity. The occurrence of motion artifacts was tested by electrical recordings of the patella. The involvement of reflex activity was investigated by analyzing the magnitude of the isolated spikes during changes in voluntary background muscle activity. Eighteen physically active volunteers performed static squats while the sEMG was measured of five lower limb muscles during vertical WBV using no load and an additional load of 33 kg. In order to record motion artifacts during WBV, a pair of electrodes was positioned on the patella with several layers of tape between skin and electrodes. Spectral analysis of the patella signal revealed recordings of motion artifacts as high peaks at the vibration frequency (fundamental and marginal peaks at the multiple harmonics were observed. For the sEMG recordings, the root mean square of the spikes increased with increasing additional loads (p < 0.05, and was significantly correlated to the sEMG signal without the spikes of the respective muscle (r range: 0.54 - 0.92, p < 0.05. This finding indicates that reflex activity might be contained in the isolated spikes, as identical behavior has been found for stretch reflex responses evoked during direct vibration. In conclusion, the spikes visible in the sEMG spectrum during WBV exercises contain motion artifacts and possibly reflex activity.

Structure-activity relationship (SAR) study and design strategies of nitrogen-containing heterocyclic moieties for their anticancer activities.

Science.gov (United States)

Akhtar, Jawaid; Khan, Ahsan Ahmed; Ali, Zulphikar; Haider, Rafi; Shahar Yar, M

2017-01-05

The present review article offers a detailed account of the design strategies employed for the synthesis of nitrogen-containing anticancer agents. The results of different studies describe the N-heterocyclic ring system is a core structure in many synthetic compounds exhibiting a broad range of biological activities. Benzimidazole, benzothiazole, indole, acridine, oxadiazole, imidazole, isoxazole, pyrazole, triazoles, quinolines and quinazolines including others drugs containing pyridazine, pyridine and pyrimidines are covered. The following studies of these compounds suggested that these compounds showed their antitumor activities through multiple mechanisms including inhibiting protein kinase (CDK, MK-2, PLK1, kinesin-like protein Eg5 and IKK), topoisomerase I and II, microtubule inhibition, and many others. Our concise representation exploits the design and anticancer potency of these compounds. The direct comparison of anticancer activities with the standard enables a systematic analysis of the structure-activity relationship among the series. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Development and Application of a New Microarray- Based Method for High-Throughput Screening of Carbohydrate Active Enzymes

DEFF Research Database (Denmark)

Vidal Melgosa, Silvia

single defined polysaccharides, mixtures of defined polysaccharides and complex biomass extracts. Furthermore, the capacity of the technique to analyse endo- and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases has been confirmed...... will contribute to both the discovery of CAZymes and the empirical characterisation of their activities, thus aiding their industrial utilisation and biological understanding...

In vitro antimicrobial activity and biocompatibility of propolis containing nanohydroxyapatite

International Nuclear Information System (INIS)

Grenho, L; Barros, J; Monteiro, F J; Ferraz, M P; Ferreira, C; Santos, V R; Cortes, M E

2015-01-01

The high number of biomaterial associated infections demands new strategies to prevent this problem. In this study the suitability of nanohydroxyapatite (nanoHA)-based surfaces containing two Brazilian extracts of propolis (green and red ones) to prevent bacterial growth and biofilm formation, as well as its non-cytotoxic nature, was investigated. Optical density, colony forming units and MTT reduction assay were used to assess the materials’ antibacterial activity against planktonic and sessile growth of Staphylococcus aureus. NanoHA matrix was able to absorb both types of propolis and the obtained results revealed the antibacterial effectiveness of the novel materials expressed as the reduction of bacterial growth and biofilm formation ability. Additionally, cell culture tests showed the growth of fibroblasts with high metabolic activity and without membrane damage. Therefore, these nanoHA-based surfaces containing natural products deriving from bees may be a promising bioactive biomaterial to be further studied with the aim of application to orthopaedic or dental devices. (paper)

77 FR 21579 - Agency Information Collection Activities: Transfer of Cargo to a Container Station

Science.gov (United States)

2012-04-10

... Activities: Transfer of Cargo to a Container Station AGENCY: U.S. Customs and Border Protection, Department... a Container Station. This is a proposed extension of an information collection that was previously... Container Station. OMB Number: 1651-0096. Form Number: None. Abstract: Before the filing of an entry of...

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

Science.gov (United States)

Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics.

Science.gov (United States)

Kadek, Alan; Kavan, Daniel; Marcoux, Julien; Stojko, Johann; Felice, Alfons K G; Cianférani, Sarah; Ludwig, Roland; Halada, Petr; Man, Petr

2017-02-01

Cellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far. To investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used. HDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation. Transition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH. The results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Possible Peroxo State of the Dicopper Site of Particulate Methane Monooxygenase from Combined Quantum Mechanics and Molecular Mechanics Calculations.

Science.gov (United States)

Itoyama, Shuhei; Doitomi, Kazuki; Kamachi, Takashi; Shiota, Yoshihito; Yoshizawa, Kazunari

2016-03-21

Enzymatic methane hydroxylation is proposed to efficiently occur at the dinuclear copper site of particulate methane monooxygenase (pMMO), which is an integral membrane metalloenzyme in methanotrophic bacteria. The resting state and a possible peroxo state of the dicopper active site of pMMO are discussed by using combined quantum mechanics and molecular mechanics calculations on the basis of reported X-ray crystal structures of the resting state of pMMO by Rosenzweig and co-workers. The dicopper site has a unique structure, in which one copper is coordinated by two histidine imidazoles and another is chelated by a histidine imidazole and primary amine of an N-terminal histidine. The resting state of the dicopper site is assignable to the mixed-valent Cu(I)Cu(II) state from a computed Cu-Cu distance of 2.62 Å from calculations at the B3LYP-D/TZVP level of theory. A μ-η(2):η(2)-peroxo-Cu(II)2 structure similar to those of hemocyanin and tyrosinase is reasonably obtained by using the resting state structure and dioxygen. Computed Cu-Cu and O-O distances are 3.63 and 1.46 Å, respectively, in the open-shell singlet state. Structural features of the dicopper peroxo species of pMMO are compared with those of hemocyanin and tyrosinase and synthetic dicopper model compounds. Optical features of the μ-η(2):η(2)-peroxo-Cu(II)2 state are calculated and analyzed with TD-DFT calculations.

One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.

Science.gov (United States)

Boerma, Jan Simon; Vermeulen, Nico P E; Commandeur, Jan N M

2014-01-25

Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2'-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The Role of Biotransformation and Oxidative Stress in 3,5-Dichloroaniline (3,5-DCA) Induced Nephrotoxicity in Isolated Renal Cortical Cells from Male Fischer 344 Rats

Science.gov (United States)

Racine, Christopher R.; Ferguson, Travis; Preston, Debbie; Ward, Dakota; Ball, John; Anestis, Dianne; Valentovic, Monica; Rankin, Gary O.

2016-01-01

Among the mono- and dichloroanilines, 3,5-Dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (~4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0 mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0 mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro. PMID:26808022

Proposed development programme for a temporary containment system for alpha active decommissioning

International Nuclear Information System (INIS)

Pengelly, M.G.A.; Burnett, R.C.

1983-06-01

This report makes a proposal to design, develop and test a containment of modular construction under plutonium active conditions. While this proposal contemplates work with plutonium, the system, when fully developed, has obvious applications wherever a temporary containment of radioactive or toxic materials is required. The fundamental feature of the proposal is that strippable coatings are used to prevent the inner surfaces of the working area from becoming contaminated. It is envisaged that this method of protecting the surfaces will enable the modular containment structure to be disassembled and re-used. (author)

Magnetic resonance studies on the copper site of dopamine β-monooxygenase in the presence of cyanide and azide anions

International Nuclear Information System (INIS)

Obata, A.; Tanaka, H.; Kawazura, H.

1987-01-01

In order to elucidate the coordination state of water molecules in the Cu(II) site of dopamine [(3,4-dihydroxyphenyl)ethylamine] β-monooxygenase, measurements of the paramagnetic 1 H nuclear magnetic relaxation rate of solvent water in the enzyme solution containing cyanide or azide as an exogenous ligand were carried out to obtain the values of intrinsic paramagnetic relaxation rate decrements R/sub p/ 1 and R/sub p/ 2 for the ligand-enzyme 1:1 and 2:1 complexes, respectively. R/sub p/ 1 (percent) values were 53 (pH 5.5) and 52 (pH 7.0) for cyanide and 38 (pH 5.5) and 32 (pH 7.0) for azide, while R/sub p/ 2 (percent) values were 98 (pH 5.5) and 96 (pH 7.0) for azide. Although no R/sub p/ 2 values for cyanide were obtained because of its reducing power at the Cu(II) site, the R/sub p/ 1 and R/sub p/ 2 values obtained above prove that the Cu(II) center has two coordinated water molecules that are exchangeable for exogenous ligands at either pH. Supporting evidence was provided by electron paramagnetic resonance (EPR) titration, in which the enzyme solution containing cyanide-enzyme (1:1) complex in an equal proportion to uncomplexed enzyme gave an observed paramagnetic relaxation rate decrement, R/sub p/, of 23%. Another characteristic of the R/sub p/ 1 and R/sub p/ 2 values was their invariability with respect to pH, indicating that the three-dimensional structure of the Cu(II) site is pH-invariant within the range examined. Binding constants of ligand to enzyme K/sub b/ 1 and K/sub b/ 2 for 1:1 and 2:1 complex formation, respectively, were also determined through an analysis of the R/sub p/ values; it was found that K/sub b/ 1 was larger than K/sub b/ 2 irrespective of pH. On the basis of these results, together with the axial-symmetric EPR parameters of the 1:1 complexes, a possible coordination geometry of the two water molecules in the Cu(II) site of the enzyme is suggested

Absence of kynurenine 3-monooxygenase reduces mortality of acute viral myocarditis in mice.

Science.gov (United States)

Kubo, Hisako; Hoshi, Masato; Mouri, Akihiro; Tashita, Chieko; Yamamoto, Yasuko; Nabeshima, Toshitaka; Saito, Kuniaki

2017-01-01

Infection of the encephalomyocarditis virus (EMCV) in mice is an established model for viral myocarditis. Previously, we have demonstrated that indoleamine 2,3-dioxygenase (IDO), an L-tryptophan - kynurenine pathway (KP) enzyme, affects acute viral myocarditis. However, the roles of KP metabolites in EMCV infection remain unclear. Kynurenine 3-monooxygenase (KMO) is one of the key regulatory enzymes, which metabolizes kynurenine to 3-hydroxykynurenine in the KP. Therefore, we examined the role of KMO in acute viral infection by comparing between KMO -/- mice and KMO +/+ mice. KMO deficiency resulted in suppressed mortality after EMCV infection. The number of infiltrating cells and F4/80 + cells in KMO -/- mice was suppressed compared with those in KMO +/+ mice. KMO -/- mice showed significantly increased levels of serum KP metabolites, and induction of KMO expression upon EMCV infection was involved in its effect on mortality through EMCV suppression. Furthermore, KMO -/- mice showed significantly suppression of CCL2, CCL3 and CCL4 on day 2 and CXCL1 on day 4 after infection. These results suggest that increased KP metabolites reduced chemokine production, resulting in suppressed mortality upon KMO knockdown in EMCV infection. KP metabolites may thus provide an effective strategy for treating acute viral myocarditis. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

Science.gov (United States)

Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

2015-02-01

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

Isolation and characterization of styrene metabolism genes from styrene-assimilating soil bacteria Rhodococcus sp. ST-5 and ST-10.

Science.gov (United States)

Toda, Hiroshi; Itoh, Nobuya

2012-01-01

Styrene metabolism genes were isolated from styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10. Strain ST-5 had a gene cluster containing four open reading frames which encoded styrene degradation enzymes. The genes showed high similarity to styABCD of Pseudomonas sp. Y2. On the other hand, strain ST-10 had only two genes which encoded styrene monooxygenase and flavin oxidoreductase (styAB). Escherichia coli transformants possessing the sty genes of strains ST-5 and ST-10 produced (S)-styrene oxide from styrene, indicating that these genes function as styrene degradation enzymes. Metabolite analysis by resting-cell reaction with gas chromatography-mass spectrometry revealed that strain ST-5 converts styrene to phenylacetaldehyde via styrene oxide by styrene oxide isomerase (styC) reaction. On the other hand, strain ST-10 lacked this enzyme, and thus accumulated styrene oxide as an intermediate. HPLC analysis showed that styrene oxide was spontaneously isomerized to phenylacetaldehyde by chemical reaction. The produced phenylacetaldehyde was converted to phenylacetic acid (PAA) in strain ST-10 as well as in strain ST-5. Furthermore, phenylacetic acid was converted to phenylacetyl-CoA by the catalysis of phenylacetate-CoA ligase in strains ST-5 and ST-10. This study proposes possible styrene metabolism pathways in Rhodococcus sp. strains ST-5 and ST-10. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Lignocellulose pretreatment technologies affect the level of enzymatic cellulose oxidation by LPMO

DEFF Research Database (Denmark)

Rodríguez-Zúñiga, Ursula Fabiola; Cannella, David; de Campos Giordano, Roberto

2015-01-01

of the cellulose oxidizing enzyme lytic polysaccharide monooxygenase (LPMO). The highest activity of LPMO was observed for the hydrothermally pretreated biomasses, which also contained the highest level of lignin. All hydrolysis were done at high dry matter levels, using a commercial enzyme preparation containing......Sugarcane bagasse, corn stover, and wheat straw are among the most available resources for production of cellulosic ethanol. For these biomasses we study the influence of pre-treatment methods on the chemical composition, as well as on the subsequent reactions of enzymatic hydrolysis and oxidation...

Genetic variants of the kynurenine-3-monooxygenase and postpartum depressive symptoms after cesarean section in Chinese women.

Science.gov (United States)

Wang, Sai-Ying; Duan, Kai-Ming; Tan, Xiao-Fang; Yin, Ji-Ye; Mao, Xiao-Yuan; Zheng, Wei; Wang, Chun-Yan; Yang, Mi; Peng, Cheng; Zhou, Hong-Hao; Liu, Zhao-Qian

2017-06-01

New conceptualizations of depression have emphasized the role of the kynurenine pathway (KP) in the pathogenesis of postpartum depressive symptoms (PDS). Kynurenine 3-monooxygenase (KMO) is a rate-limiting enzyme of the KP, where it catalyzes the conversion of kynurenine (KYN) to 3-hydroxykynurenine (3-HK). Previous work indicates that KMO is closely linked to the pathophysiology of depressive disorders. The purpose of this study is to investigate whether variations in the KMO gene affect PDS development after cesarean section. A total of 710 Chinese women receiving cesarean section were enrolled in this study. PDS was determined by an Edinburgh Postnatal Depression Scale (EPDS) score ≥13. Subsequently, 24 women with PDS and 48 matched women without PDS were randomly selected for investigation of perinatal serum concentrations of KYN, 3-HK and the 3-HK/KYN ratio. The 3-HK/KYN ratio indicates the activity of KMO. In addition, 6 single nucleotide polymorphisms of the KMO gene were examined. Following this genotyping, 36 puerperant women carrying the KMO rs1053230 AG genotype and 72 matched puerperant women carrying the KMO rs1053230 GG genotype were selected for comparisons of KYN, 3-HK and 3-HK/KYN ratio levels. The results show the incidence of PDS in the Chinese population to be 7.3%, with PDS characterized by increased serum 3-HK concentration and 3-HK/KYN ratio, versus matched postpartum women without PDS (PKMO rs1053230 are significantly associated with the incidence of PDS (PKMO rs1053230 AG genotype are significantly higher than those in matched postpartum women carrying the KMO rs1053230 GG genotype. The presented data highlight the contribution of alterations in the KP to the pathogenesis of postpartum depression. Heightened KMO activity, including as arising from KMO rs1053230 G/A genetic variations, are indicated as one possible mechanism driving the biological underpinnings of PDS. Copyright © 2017 Elsevier B.V. All rights reserved.

Activity of molybdenum-containing oxide catalysts in the reaction of ethane oxidation

International Nuclear Information System (INIS)

Konovalov, V.I.; Ehpova, T.I.; Shchukin, V.P.; Averbukh, A.Ya.

1977-01-01

Investigation results concerning the catalytic activity of molybdenum-containing catalysts in ethane oxidation reaction are presented. It has been found that the greatest activity in the temperature range from 450 to 600 deg C is exhibited by cobalt-molybdenum catalyst; at 600 deg C bismuth-molybdenum catalyst is the most active. Nickel-molybdenum catalyst is selective and active with respect to ethylene. Iron- and manganese-molybdenum catalysts do not show high ethane oxidation rates and their selectivity is insignificant

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

Science.gov (United States)

Bass, C; Carvalho, R A; Oliphant, L; Puinean, A M; Field, L M; Nauen, R; Williamson, M S; Moores, G; Gorman, K

2011-12-01

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

Cytochrome P450 genes from the aquatic midge Chironomus tentans: Atrazine-induced up-regulation of CtCYP6EX3 contributing to oxidative activation of chlorpyrifos

Science.gov (United States)

The open reading frames of 19 cytochrome P450 monooxygenase (CYP) genes were sequenced from Chironomus tentans, a commonly used freshwater invertebrate model. Functional analysis of CtCYP6EX3 confirmed its atrazine-induced oxidative activation for chlorpyrifos by using a nanoparticle-based RNA inter...

Imidazole-containing farnesyltransferase inhibitors: 3D quantitative structure-activity relationships and molecular docking

Science.gov (United States)

Xie, Aihua; Odde, Srinivas; Prasanna, Sivaprakasam; Doerksen, Robert J.

2009-07-01

One of the most promising anticancer and recent antimalarial targets is the heterodimeric zinc-containing protein farnesyltransferase (FT). In this work, we studied a highly diverse series of 192 Abbott-initiated imidazole-containing compounds and their FT inhibitory activities using 3D-QSAR and docking, in order to gain understanding of the interaction of these inhibitors with FT to aid development of a rational strategy for further lead optimization. We report several highly significant and predictive CoMFA and CoMSIA models. The best model, composed of CoMFA steric and electrostatic fields combined with CoMSIA hydrophobic and H-bond acceptor fields, had r 2 = 0.878, q 2 = 0.630, and r pred 2 = 0.614. Docking studies on the statistical outliers revealed that some of them had a different binding mode in the FT active site based on steric bulk and available active site space, explaining why the predicted activities differed from the experimental activities.

CZECHOSLOVAK ACTIVITY TO PREPARE EUROPEAN NORMS FOR CONTAINERS BEFORE THE SECOND WORLD WAR

Directory of Open Access Journals (Sweden)

Krzysztof Lewandowski

2014-12-01

Full Text Available In many articles we can read that containers weren't in Europe before 1966, when ship SS Fariland came with 35 feet containers invented by Keith Tantlinger for Sea Land Company owned by Malcom McLean. The focus of this study is on the problem with development norms for European containers. Thus, the main definitions and briefly literature overview in the analysed research area are given. Later, the information about these constructions are developed. Article presents Czechoslovak activity to preparation of three European norms for containers, which were described before Second World War.

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Science.gov (United States)

2012-11-20

... Activities: Holders or Containers Which Enter the United States Duty Free AGENCY: U.S. Customs and Border... information collection requirement concerning the Holders or Containers which Enter the United States Duty... concerning the following information collection: Title: Holders or Containers which Enter the United States...

Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1*

Science.gov (United States)

Baron, Daniel; LaBelle, Edward; Coursolle, Dan; Gralnick, Jeffrey A.; Bond, Daniel R.

2009-01-01

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at ∼0 V (versus standard hydrogen electrode), and was altered in single (ΔomcA, ΔmtrC) and double deletion (ΔomcA/ΔmtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (−0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (∼1 s−1). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies. PMID:19661057

Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria ▿

Science.gov (United States)

Furuya, Toshiki; Hirose, Satomi; Semba, Hisashi; Kino, Kuniki

2011-01-01

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ54 factor, a gene encoding the σ54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. PMID:21856847

78 FR 9720 - Agency Information Collection Activities: Holders or Containers Which Enter the United States...

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2013-02-11

... Activities: Holders or Containers Which Enter the United States Duty Free AGENCY: U.S. Customs and Border... or Containers which enter the United States Duty Free. This is a proposed extension of an information..., or other technological techniques or other forms of information. Title: Holders or Containers which...

Antibacterial and antifungal activity of sulfur-containing compounds from Petiveria alliacea L.

Science.gov (United States)

Kim, Seokwon; Kubec, Roman; Musah, Rabi A

2006-03-08

A total of 18 organosulfur compounds originating from Petiveria alliacea L. roots have been tested for their antibacterial and antifungal activities. These represent compounds occurring in fresh homogenates as well as those present in various macerates, extracts and other preparations made from Petiveria alliacea. Of the compounds assayed, the thiosulfinates, trisulfides and benzylsulfinic acid were observed to be the most active, with the benzyl-containing thiosulfinates exhibiting the broadest spectrum of antimicrobial activity. The effect of plant sample preparation conditions on the antimicrobial activity of the extract is discussed.

Microscopic and spectroscopic properties of Langmuir–Blodgett films composed of flavins and their aggregation structures

International Nuclear Information System (INIS)

Lim, Jong Kuk; Jo, Jihee; Jang, Dasol; Jang, Hyeong Ju

2015-01-01

Isoalloxazine derivatives (flavins) are commonly found in natural systems that are involved in an electron transfer process, such as photosynthetic or metabolic systems, and are also frequently used as electron donors in organic-based electronic devices. As an example, molecular photodiodes composed of 7,8-dimethyl-10-dodecyl isoalloxazine (DDI) have been fabricated by the Langmuir–Blodgett (LB) technique, and such devices showed characteristic properties of photodiodes. The efficiency of molecular photodiodes is dependent on the assembled structure of the LB films, which is related to the morphology of the LB films. For that reason, Lim has investigated the morphology of LB films, and found that rod-shaped domains are formed when a DDI monolayer is transferred to a solid substrate above a specific surface pressure (Thin Solid Films, 531 (2013) 499). In that paper, rod-shaped domains were revealed to be collapsed triple layers, i.e., double layers collapsed on the monolayer; however, the detailed aggregation structure of the constituent molecules (DDI) has not been studied. Herein, we investigate the microscopic and spectroscopic properties of LB films composed of DDI. We apply the extended dipole model to explain spectral changes in the absorption spectra and propose an aggregation structure for DDI in the LB films. - Highlights: • Aggregation structure of DDI in LB films was experimentally investigated. • Theoretical estimation is in good agreement with experimental result. • Molecular aggregation structure for DDI in LB films was proposed. • Molecular configuration in LB films is changed from side-by-side to face-to-face.

Shatavarins (containing Shatavarin IV) with anticancer activity from the roots of Asparagus racemosus

Science.gov (United States)

Mitra, Shankar K.; Prakash, Neswi S.; Sundaram, Ramachandran

2012-01-01

Objectives: The anticancer activity of shatavarins (containing shatavarin IV) isolated from the roots of Asparagus racemosus (Wild) was evaluated using in vitro and in vivo experimental models. Material and Methods: The shatavarin IV was isolated from ethyl acetate insoluble fraction (AR-2B) of chloroform:methanol (2:1) (AR-2) extract of A. racemosus roots. The cytotoxicity (in vitro) of shatavarin IV and other shatavarins rich fraction was carried out using of MTT assay using MCF-7 (human breast cancer), HT-29 (human colon adenocarcinoma), and A-498 (human kidney carcinoma) cell lines. The in vivo anticancer activity of shatavarins (containing shatavarin IV) was evaluated against Ehrlich ascites carcinoma (EAC) tumor bearing mice. Results: The isolated shatavarin IV (84.69 %) along with shatavarins rich fraction, coded AR-2B containing 5.05% shatavarin IV showed potent cytotoxicity. Oral administration of AR-2B to tumor bearing mice at doses of 250 and 500 mg/kg body weight for 10 days, showed significant reduction in percent increase in body weight, tumor volume, packed cell volume, viable tumor cell count, and increased non-viable cell count when compared to the untreated mice of the EAC control group. The restoration of hematological parameters towards normalcy was also observed. Conclusion: The result suggests that the shatavarins (containing shatavarin IV) rich fraction (AR-2B) exhibits significant anticancer activity in both in vitro and in vivo experimental models. PMID:23248403

Alternative containers for low-level wastes containing large amounts of tritium

International Nuclear Information System (INIS)

Gause, E.P.; Lee, B.S.; MacKenzie, D.R.; Wiswall, R. Jr.

1984-11-01

High-activity tritiated waste generated in the United States is mainly composed of tritium gas and tritium-contaminated organic solvents sorbed onto Speedi-Dri which are packaged in small glass bulbs. Low-activity waste consists of solidified and adsorbed liquids. In this report, current packages for high-activity gaseous and low-activity adsorbed liquid wastes are emphasized with regard to containment potential. Containers for low-level radioactive waste containing large amounts of tritium need to be developed. An integrity may be threatened by: physical degradation due to soil corrosion, gas pressure build-up (due to radiolysis and/or biodegradation), rapid permeation of tritium through the container, and corrosion from container contents. Literature available on these points is summarized in this report. 136 references, 20 figures, 40 tables

Tryptophan-Containing Cyclic Decapeptides with Activity against Plant Pathogenic Bacteria

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Cristina Camó

2017-10-01

Full Text Available A library of 66 cyclic decapeptides incorporating a Trp residue was synthesized on solid phase and screened against the phytopathogenic bacteria Pseudomonas syringae pv. syringae, Xanthomonas axonopodis pv. vesicatoria, and Erwinia amylovora. The hemolytic activity of these peptides was also evaluated. The results obtained were compared with those of a collection of Phe analogues previously reported. The analysis of the data showed that the presence of the Trp improved the antibacterial activity against these three pathogens. In particular, 40 to 46 Trp analogues displayed lower minimum inhibitory concentration (MIC values than their corresponding Phe counterparts. Interestingly, 26 Trp-containing sequences exhibited MIC of 0.8 to 3.1 μM against X. axonopodis pv. vesicatoria, 21 peptides MIC of 1.6 to 6.2 μM against P. syringae pv. syringae and six peptides MIC of 6.2 to 12.5 μM against E. amylovora. Regarding the hemolysis, in general, Trp derivatives displayed a percentage of hemolysis comparable to that of their Phe analogues. Notably, 49 Trp-containing cyclic peptides showed a hemolysis ≤ 20% at 125 μM. The peptides with the best biological activity profile were c(LKKKLWKKLQ (BPC086W and c(LKKKKWLLKQ (BPC108W, which displayed MIC values ranging from 0.8 to 12.5 μM and a hemolysis ≤ 8% at 125 μM. Therefore, it is evident that these Trp sequences constitute promising candidates for the development of new agents for use in plant protection.

Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

Directory of Open Access Journals (Sweden)

Paul P. Kelly

2015-09-01

Full Text Available Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

Synthesis and fungicidal activity of pyrazole derivatives containing 1,2,3,4-tetrahydroquinoline.

Science.gov (United States)

Lei, Peng; Zhang, Xuebo; Xu, Yan; Xu, Gaofei; Liu, Xili; Yang, Xinling; Zhang, Xiaohe; Ling, Yun

2016-01-01

Take-all of wheat, caused by the soil-borne fungus Gaeumannomyces graminis var. tritici, is one of the most important and widespread root diseases. Given that take-all is still hard to control, it is necessary to develop new effective agrochemicals. Pyrazole derivatives have been often reported for their favorable bioactivities. In order to discover compounds with high fungicidal activity and simple structures, 1,2,3,4-tetrahydroquinoline, a biologically active group of natural products, was introduced to pyrazole structure. A series of pyrazole derivatives containing 1,2,3,4-tetrahydroquinoline were synthesized, and their fungicidal activities were evaluated. The bioassay results demonstrated that the title compounds displayed obvious fungicidal activities at a concentration of 50 μg/mL, especially against V. mali, S. sclerotiorum and G. graminis var. tritici. The inhibition rates of compounds 10d, 10e, 10h, 10i and 10j against G. graminis var. tritici were all above 90 %. Even at a lower concentration of 16.7 μg/mL, compounds 10d and 10e exhibited satisfied activities of 100 % and 94.0 %, respectively. It is comparable to that of the positive control pyraclostrobin with 100 % inhibition rate. A series of pyrazole derivatives containing 1,2,3,4-tetrahydroquinoline were synthesized and their structures were confirmed by (1)H NMR, (13)C NMR, IR spectrum and HRMS or elemental analysis. The crystal structure of compound 10g was confirmed by X-ray diffraction. Bioassay results indicated that all title compounds exhibited obvious fungicidal activities. In particular, compounds 10d and 10e showed comparable activities against G. graminis var. tritici with the commercial fungicide pyraclostrobin at the concentration of 16.7 μg/mL.Graphical abstractA series of pyrazole derivatives containing 1,2,3,4-tetrahydroquinoline were designed and synthesized. Bioassay results indicated that all these compounds exhibited obvious fungicidal activities.

Microparticles Containing Curcumin Solid Dispersion: Stability, Bioavailability and Anti-Inflammatory Activity.

Science.gov (United States)

Teixeira, C C C; Mendonça, L M; Bergamaschi, M M; Queiroz, R H C; Souza, G E P; Antunes, L M G; Freitas, L A P

2016-04-01

This work aimed at improving the solubility of curcumin by the preparation of spray-dried ternary solid dispersions containing Gelucire®50/13-Aerosil® and quantifying the resulting in vivo oral bioavailability and anti-inflammatory activity. The solid dispersion containing 40% of curcumin was characterised by calorimetry, infrared spectroscopy and X-ray powder diffraction. The solubility and dissolution rate of curcumin in aqueous HCl or phosphate buffer improved up to 3600- and 7.3-fold, respectively. Accelerated stability test demonstrated that the solid dispersion was stable for 9 months. The pharmacokinetic study showed a 5.5-fold increase in curcumin in rat blood plasma when compared to unprocessed curcumin. The solid dispersion also provided enhanced anti-inflammatory activity in rat paw oedema. Finally, the solid dispersion proposed here is a promising way to enhance curcumin bioavailability at an industrial pharmaceutical perspective, since its preparation applies the spray drying, which is an easy to scale up technique. The findings herein stimulate further in vivo evaluations and clinical tests as a cancer and Alzheimer chemoprevention agent.

Dreissenid mussels from the Great Lakes contain elevated thiaminase activity

Science.gov (United States)

Tillitt, D.E.; Riley, S.C.; Evans, A.N.; Nichols, S.J.; Zajicek, J.L.; Rinchard, J.; Richter, C.A.; Krueger, C.C.

2009-01-01

We examined thiaminase activity in dreissenid mussels collected at different depths and seasons, and from various locations in Lakes Michigan, Ontario, and Huron. Here we present evidence that two dreissenid mussel species (Dreissena bugensis and D. polymorpha) contain thiaminase activity that is 5-100 fold greater than observed in Great Lakes fishes. Thiaminase activity in zebra mussels ranged from 10,600 to 47,900??pmol g- 1??min- 1 and activities in quagga mussels ranged from 19,500 to 223,800??pmol g- 1??min- 1. Activity in the mussels was greatest in spring, less in summer, and least in fall. Additionally, we observed greater thiaminase activity in dreissenid mussels collected at shallow depths compared to mussels collected at deeper depths. Dreissenids constitute a significant and previously unknown pool of thiaminase in the Great Lakes food web compared to other known sources of this thiamine (vitamin B1)-degrading enzyme. Thiaminase in forage fish of the Great Lakes has been causally linked to thiamine deficiency in salmonines. We currently do not know whether linkages exist between thiaminase activities observed in dreissenids and the thiaminase activities in higher trophic levels of the Great Lakes food web. However, the extreme thiaminase activities observed in dreissenids from the Great Lakes may represent a serious unanticipated negative effect of these exotic species on Great Lakes ecosystems.

Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials.

Science.gov (United States)

Zafar, Muhammad Nadeem; Wang, Xiaoju; Sygmund, Christoph; Ludwig, Roland; Leech, Dónal; Gorton, Lo

2012-01-03

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration. © 2011 American Chemical Society

A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes

DEFF Research Database (Denmark)

Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia

2015-01-01

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolases...... that the technique can be used to analyse both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified un-characterised enzymes...

The lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

Energy Technology Data Exchange (ETDEWEB)

Otoikhian, Adenike [Oregon Health & Sciences University; Barry, Amanda N. [Los Alamos National Laboratory; Mayfield, Mary [Oregon Health & Science University; Nilges, Mark [Illinois EPR Center; Huang, Yiping [Johns Hopkins University; Lutsenko, Svetlana [Johns Hopkins University; Blackburn, Ninian [Oregon Health & Science University

2012-05-14

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis.

Science.gov (United States)

Lobo, Leandro A; Smith, Charles J; Rocha, Edson R

2011-04-01

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

Energy Technology Data Exchange (ETDEWEB)

O; Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.; Meilleur, Flora (ORNL); (NCSU)

2017-01-19

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

Mark II containment program load evaluation and acceptance criteria; Generic Technical Activity A-8

International Nuclear Information System (INIS)

Anderson, C.J.

1981-08-01

The report provides a discussion of LOCA-related suppression pool hydrodynamic loads in boiling water reactor (BWR) facilities with the Mark II pressure-suppression containment design. This report concludes NRC Generic Technical Activity A-8, 'Mark II Containment Pool Dynamic Loads,' which has been designated an 'Unresolved Safety Issue' pursuant to Section 210 of the Energy Reorganization Act of 1974

A study on the improved DTC method for estimations of radionuclide activity in radwaste containers

International Nuclear Information System (INIS)

Kang, Sang Hee; Hwang, Ki Ha; Lee, Sang Chul; Lee, Kun Jai; Kim, Tae Wook; Kim, Kyoung Deok; Herr, Young Hoi; Song, Myung Jae

2004-01-01

Disposal of rad waste containers requires the assessment of the radioactive contents of each container. Some containers can not be assessed by the γ nuclide analyzer because of time constraint and economical burden. One alternative method, dose to curie conversion (DTC) method can provide an estimate of the container activity. This study evaluates the impact of voids, the chemical composition and density of the material and the distribution of the source related to surface dose rate and the development of the improved DTC method for more accurate assessment

A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor.

Science.gov (United States)

Vishwanatha, Kurutihalli; Bäck, Nils; Mains, Richard E; Eipper, Betty A

2014-05-02

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice.

Science.gov (United States)

Luo, Anding; Qian, Qian; Yin, Hengfu; Liu, Xiaoqiang; Yin, Changxi; Lan, Ying; Tang, Jiuyou; Tang, Zuoshun; Cao, Shouyun; Wang, Xiujie; Xia, Kai; Fu, Xiangdong; Luo, Da; Chu, Chengcai

2006-02-01

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.

Synchronous Parallel Emulation and Discrete Event Simulation System with Self-Contained Simulation Objects and Active Event Objects

Science.gov (United States)

Steinman, Jeffrey S. (Inventor)

1998-01-01

The present invention is embodied in a method of performing object-oriented simulation and a system having inter-connected processor nodes operating in parallel to simulate mutual interactions of a set of discrete simulation objects distributed among the nodes as a sequence of discrete events changing state variables of respective simulation objects so as to generate new event-defining messages addressed to respective ones of the nodes. The object-oriented simulation is performed at each one of the nodes by assigning passive self-contained simulation objects to each one of the nodes, responding to messages received at one node by generating corresponding active event objects having user-defined inherent capabilities and individual time stamps and corresponding to respective events affecting one of the passive self-contained simulation objects of the one node, restricting the respective passive self-contained simulation objects to only providing and receiving information from die respective active event objects, requesting information and changing variables within a passive self-contained simulation object by the active event object, and producing corresponding messages specifying events resulting therefrom by the active event objects.

Genome-wide association identifies genetic variants associated with lentiform nucleus volume in N = 1345 young and elderly subjects.

Science.gov (United States)

Hibar, Derrek P; Stein, Jason L; Ryles, April B; Kohannim, Omid; Jahanshad, Neda; Medland, Sarah E; Hansell, Narelle K; McMahon, Katie L; de Zubicaray, Greig I; Montgomery, Grant W; Martin, Nicholas G; Wright, Margaret J; Saykin, Andrew J; Jack, Clifford R; Weiner, Michael W; Toga, Arthur W; Thompson, Paul M

2013-06-01

Deficits in lentiform nucleus volume and morphometry are implicated in a number of genetically influenced disorders, including Parkinson's disease, schizophrenia, and ADHD. Here we performed genome-wide searches to discover common genetic variants associated with differences in lentiform nucleus volume in human populations. We assessed structural MRI scans of the brain in two large genotyped samples: the Alzheimer's Disease Neuroimaging Initiative (ADNI; N = 706) and the Queensland Twin Imaging Study (QTIM; N = 639). Statistics of association from each cohort were combined meta-analytically using a fixed-effects model to boost power and to reduce the prevalence of false positive findings. We identified a number of associations in and around the flavin-containing monooxygenase (FMO) gene cluster. The most highly associated SNP, rs1795240, was located in the FMO3 gene; after meta-analysis, it showed genome-wide significant evidence of association with lentiform nucleus volume (P MA  = 4.79 × 10(-8)). This commonly-carried genetic variant accounted for 2.68 % and 0.84 % of the trait variability in the ADNI and QTIM samples, respectively, even though the QTIM sample was on average 50 years younger. Pathway enrichment analysis revealed significant contributions of this gene to the cytochrome P450 pathway, which is involved in metabolizing numerous therapeutic drugs for pain, seizures, mania, depression, anxiety, and psychosis. The genetic variants we identified provide replicated, genome-wide significant evidence for the FMO gene cluster's involvement in lentiform nucleus volume differences in human populations.

Interspecies Variation of In Vitro Stability and Metabolic Diversity of YZG-331, a Promising Sedative-Hypnotic Compound

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Zhihao Liu

2017-08-01

Full Text Available YZG-331, a synthetic adenosine derivative, express the sedative and hypnotic effects via binding to the adenosine receptor. The current study was taken to investigate the metabolic pathway of YZG-331 as well as species-specific differences in vitro. YZG-331 was reduced by 14, 11, 6, 46, and 11% within 120 min incubation in human, monkey, dog, rat, and mouse liver microsomes (LMs, respectively. However, YZG-331 was stable in human, monkey, dog, rat, and mouse liver cytoplasm. In addition, YZG-331 was unstable in rat or mouse gut microbiota with more than 50% of prototype drug degraded within 120 min incubation. Interestingly, the systemic exposure of M2 and M3 in rats and mice treated with antibiotics were significantly decreased in the pseudo germ-free group. YZG-331 could be metabolized in rat and human liver under the catalysis of CYP enzymes, and the metabolism showed species variation. In addition, 3 phase I metabolites were identified via hydroxyl (M1, hydrolysis (M2, or hydrolysis/ hydroxyl (M3 pathway. Flavin-containing monooxygenase 1 (FMO1 and FMO3 participated in the conversion of YZG-331 in rat LMs. Nevertheless, YZG-331 expressed stability with recombinant human FMOs, which further confirmed the species variation in the metabolism. Overall, these studies suggested that YZG-331 is not stable in LMs and gut microbiota. CYP450 enzymes and FMOs mediated the metabolism of YZG-331, and the metabolic pathway showed species difference. Special attention must be paid when extrapolating data from other species to humans.

GenBank blastx search result: AK061794 [KOME

Lifescience Database Archive (English)

Full Text Available AK061794 001-039-F11 AF031161.1 Pseudomonas sp. VLB120 styrene degradation genes in...cluding histidine kinase (stdSc) gene, partial cds; and transcriptional activator (stdR), styrene monooxygen...ase large component (stdA), styrene monooxygenase small component (stdB), styrene oxide isomerase (stdC), an

A decaying factor accounts for contained activity in neuronal networks with no need of hierarchical or modular organization

International Nuclear Information System (INIS)

Amancio, Diego R; Oliveira Jr, Osvaldo N; Costa, Luciano da F

2012-01-01

The mechanisms responsible for containing activity in systems represented by networks are crucial in various phenomena, for example, in diseases such as epilepsy that affect the neuronal networks and for information dissemination in social networks. The first models to account for contained activity included triggering and inhibition processes, but they cannot be applied to social networks where inhibition is clearly absent. A recent model showed that contained activity can be achieved with no need of inhibition processes provided that the network is subdivided into modules (communities). In this paper, we introduce a new concept inspired in the Hebbian theory, through which containment of activity is achieved by incorporating a dynamics based on a decaying activity in a random walk mechanism preferential to the node activity. Upon selecting the decay coefficient within a proper range, we observed sustained activity in all the networks tested, namely, random, Barabási–Albert and geographical networks. The generality of this finding was confirmed by showing that modularity is no longer needed if the dynamics based on the integrate-and-fire dynamics incorporated the decay factor. Taken together, these results provide a proof of principle that persistent, restrained network activation might occur in the absence of any particular topological structure. This may be the reason why neuronal activity does not spread out to the entire neuronal network, even when no special topological organization exists. (paper)

Yucca Mountain project container fabrication, closure and non-destructive evaluation development activities

International Nuclear Information System (INIS)

Russell, E.W.; Nelson, T.A.

1989-06-01

In this presentation, container fabrication, closure, and non-destructive evaluation (NDE) process development activities are described. All of these activities are interrelated, and will contribute to the metal barrier selection activity. The plan is to use a corrosion-resistant material in the form of a cylinder with a wall thickness of ∼1cm (2cm for pure copper.) The materials under consideration include the three austenitic alloys: stainless steel-304L, stainless steel-316L and alloy 825, as well as the three copper alloys: CDA 102, CDA 613, and CDA 715. This document reviews the recommended procedures and processes for fabricating, closing and evaluating each of the candidate materials

Metal-containing Complexes of Lactams, Imidazoles, and Benzimidazoles and Their Biological Activity

Science.gov (United States)

Kukalenko, S. S.; Bovykin, B. A.; Shestakova, S. I.; Omel'chenko, A. M.

1985-07-01

The results of the latest investigations of the problem of the synthesis of metal-containing complexes of lactams, imidazoles, and benzimidazoles, their structure, and their stability in solutions are surveyed. Some data on their biological activity (pesticide and pharmacological) and the mechanism of their physiological action are presented. The bibliography includes 190 references.

High Efficient Enrichment and Activated Dissolution of Refractory Low Grade Rh-containing Material

Institute of Scientific and Technical Information of China (English)

WU Xiaofeng; DONG Haigang; TONG Weifeng; ZHAO Jiachun; ZENG Rui

2012-01-01

Aiming to the low-grade rhodium-containing waste materials,a new process was proposed to enrich and activate rhodium by smelting using iron oxide as a trapping agent and activator.A rhodium concentrate was obtained by the separation of base metals and precious metals.The concentrate was reacted with dilute aqua regia to obtain rhodium solution.The factors influencing the enrichment and activation effects were discussed in this paper.The results showed that the dissolution rate is greater than 99％ under the optimum conditions.In this process,the activation of rhodium was finished in the enrichment process.The iron oxide is both a trapping agent and activator,which simplifies the process and reduce the cost.

Efficacy of HOCl scavenging by sulfur-containing compounds: antioxidant activity of glutathione disulfide?

NARCIS (Netherlands)

den Hartog, G.J.M.; Haenen, G.R.M.M.; Vegt, E.; van der Vijgh, W.J.F.; Bast, A.

2002-01-01

Efficacy of HOCl scavenging by sulfur-containing compounds: antioxidant activity of glutathione disulfide? den Hartog GJ, Haenen GR, Vegt E, van der Vijgh WJ, Bast A. Department of Pharmacology and Toxicology, Maastricht University, The Netherlands. Hypochlorous acid (HOCl) is a bactericidal

System for sampling active solutions in transport container; Systeme de prelevements de solutions actives sur les recipients de transport

Energy Technology Data Exchange (ETDEWEB)

Fradin, J.

1958-12-03

This report presents a system aimed at sampling active solution from a specific transport container (SCRGR model) while transferring this solution with a maximum safety. The sampling principle is described (a flexible tube connected to the receiving container, with a needle at the other end which goes through a rubber membrane and enters a plunger tube). Its benefits are outlined (operator protection, reduction of contamination risk; only the rubber membrane is removed and replaced). Some manufacturing details are described concerning the membrane and the cover.

Vancomycin analogues containing monosaccharides exhibit improved antibiotic activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy.

Science.gov (United States)

Thayer, Desiree A; Wong, Chi-Huey

2006-09-18

Many natural products contain carbohydrate moieties that contribute to their biological activity. Manipulation of the carbohydrate domain of natural products through multiple glycosylations to identify new derivatives with novel biological activities has been a difficult and impractical process. We report a practical one-pot enzymatic approach with regeneration of cosubstrates to synthesize analogues of vancomycin that contain an N-alkyl glucosamine, which exhibited marked improvement in antibiotic activity against a vancomycin-resistant strain of Enterococcus.

Metagenomic identification of a novel salt tolerance gene from the human gut microbiome which encodes a membrane protein with homology to a brp/blh-family β-carotene 15,15'-monooxygenase.

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Eamonn P Culligan

Full Text Available The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.

Predicting the effects of microbial activity on the corrosion of copper nuclear fuel waste disposal containers

International Nuclear Information System (INIS)

King, F.; Stroes-Gascoyne, S.

1996-08-01

Microbially influenced corrosion (MIC) of copper nuclear fuel waste containers may occur in a disposal vault located 500-1000 m underground in the granitic rock of the Canadian Shield. The extent and diversity of microbial activity in the vault is expected to be limited initially because of the aggressive conditions produced by γ-radiation, elevated temperatures and desiccation of the clay-based buffer in which the containers will be embedded. Experimental results on the heat- and radiation-sensitivity of the natural microbiota in buffer material are presented. The data suggest that the low water activity in the buffer material will severely limit the growth of microbes near the container. The most likely form of MIC involves sulphate-reducing bacteria (SRB). Electrochemical experiments using a clay-covered copper electrode have shown that sulphide ions produced by SRB could diffuse through buffer material and induce corrosion of the container. A method to predict the long-term corrosion behaviour is presented. (author)

Sulfur-Containing Agrochemicals.

Science.gov (United States)

Devendar, Ponnam; Yang, Guang-Fu

2017-10-09

Modern agricultural chemistry has to support farmers by providing innovative agrochemicals. In this context, the introduction of sulfur atoms into an active ingredient is still an important tool in modulating the properties of new crop-protection compounds. More than 30% of today's agrochemicals contain at least one sulfur atom, mainly in fungicides, herbicides and insecticides. A number of recently developed sulfur-containing agrochemical candidates represent a novel class of chemical compounds with new modes of action, so we intend to highlight the emerging interest in commercially active sulfur-containing compounds. This chapter gives a comprehensive overview of selected leading sulfur-containing pesticidal chemical families namely: sulfonylureas, sulfonamides, sulfur-containing heterocyclics, thioureas, sulfides, sulfones, sulfoxides and sulfoximines. Also, the most suitable large-scale synthetic methods of the recently launched or provisionally approved sulfur-containing agrochemicals from respective chemical families have been highlighted.

Asp170 is crucial for the redox properties of vanillyl-alcohol oxidase

NARCIS (Netherlands)

Heuvel, van den R.H.H.; Fraaije, M.W.; Mattevi, A.; Berkel, van W.J.H.

2000-01-01

Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near

Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase

NARCIS (Netherlands)

Heuvel, Robert H.H. van den; Fraaije, Marco W.; Mattevi, Andrea; Berkel, Willem J.H. van

2000-01-01

Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near

The kynurenine pathway is activated in human obesity and shifted toward kynurenine monooxygenase activation.

Science.gov (United States)

Favennec, Marie; Hennart, Benjamin; Caiazzo, Robert; Leloire, Audrey; Yengo, Loïc; Verbanck, Marie; Arredouani, Abdelilah; Marre, Michel; Pigeyre, Marie; Bessede, Alban; Guillemin, Gilles J; Chinetti, Giulia; Staels, Bart; Pattou, François; Balkau, Beverley; Allorge, Delphine; Froguel, Philippe; Poulain-Godefroy, Odile

2015-10-01

This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue. Tryptophan and KP metabolite levels were measured in serum of individuals from the D.E.S.I.R. cohort (case-cohort study: 212 diabetic, 836 randomly sampled) and in women with obesity, diabetic or normoglycemic, from the ABOS cohort (n = 100). KP enzyme gene expressions were analyzed in omental and subcutaneous adipose tissue of women from the ABOS cohort, in human primary adipocytes and in monocyte-derived macrophages. In the D.E.S.I.R. cohort, kynurenine levels were positively associated with body mass index (BMI) (P = 4.68 × 10(-19) ) and with a higher HOMA2-IR insulin resistance index (P = 6.23 × 10(-4) ). The levels of kynurenine, kynurenic acid, and quinolinic acid were associated with higher BMI (P KMO], and kynurenine aminotransferase III [CCBL2]) was increased in the omental adipose tissue of women with obesity compared to lean (P KMO that is not expressed in these cells. The expressions of IDO1, KYNU, KMO, and CCBL2 were higher in proinflammatory than in anti-inflammatory macrophages (P KMO activation. © 2015 The Obesity Society.

Quantitative determination of sage-leaved rockrose (Cistus salviifolius flavonoids

Directory of Open Access Journals (Sweden)

H. M. Elkaib

2017-01-01

Full Text Available The widespread use of bioflavonoids, due to their antimicrobial activity and antioxidant properties, necessitates their separate determination in a joint presence. It is noted that the total content of flavonoids, determined by different spectrophotometric methods in the same samples, differs and depends on the structure of the individual flavonoids that make up the samples. The paper presents the results of studies to determine the content of flavonoids in the fraction of the dry extract of shalfeeloid canthus (Cistus salviifolius, which has antimicrobial activity against bacteria of the genus Pseudomonas, which causes damage to protein-containing food products. Curing flavonoids by sequential extraction with aqueous alcohol solutions, filtering and dissolving the dry residue in isopropyl alcohol followed by passing through the gel layers. The following methods for the determination of flavonoids are considered: reaction with Folin-Chikalteu reagent (using quercetin as standard, reaction with aluminum chloride and 2,4-dinitrophenylhydrazine. When oxidized in alkaline medium by the Folin-Chikalteu reagent, which is the phospho-molybdotungstate heteropolycomplexes of flavonols, flavin-3-ol and flavones can be converted into quinoid compounds having absorption bands in the visible region of the spectrum. Only flavones and flavones having hydroxyl groups at positions 3 and 5 can interact with AlCl3. 2,4-dinitrophenylhydrazine only interacts with flavonols. The obtained calibration curves and structural formulas of flavonoids are given, which can be determined by the methods presented. It was found to contain flavonols 0.21 mg / mg, flavones 0.17 mg / mg and flavin-3-ol 0.06 mg/mg.

A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase

International Nuclear Information System (INIS)

Lyubimov, Artem Y.; Chen, Lin; Sampson, Nicole S.; Vrielink, Alice

2009-01-01

The importance of active-site electrostatics for oxidative and reductive half-reactions in a redox flavoenzyme (cholesterol oxidase) have been investigated by a combination of biochemistry and atomic resolution crystallography. A detailed examination of active-site dynamics demonstrates that the oxidation of substrate and the re-oxidation of the flavin cofactor by molecular oxygen are linked by a single active-site asparagine. Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3β-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH⋯π interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall k cat . Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0 Å resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH⋯π interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered

Photodynamics of the small BLUF protein BlrB from Rhodobacter sphaeroides.

Science.gov (United States)

Zirak, P; Penzkofer, A; Schiereis, T; Hegemann, P; Jung, A; Schlichting, I

2006-06-01

The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB(I)) and recombinant BlrB containing only FAD (called BlrB(II)) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF(r,f) and BLUF(r,sl)). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF(s,f) and BLUF(s,sl)) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2s. Quantum yields of signalling state formation of about 90% for BlrB(II) and about 40% for BlrB(I) were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.

Lead corrosion evaluation in high activity nuclear waste container (Argentina)

International Nuclear Information System (INIS)

Guasp, R.; Lanzani, L.; Bruzzoni, P.; Cufre, W.; Semino, C.J.

2000-01-01

This report describes a study of high activity nuclear waste canister corrosion in a deep geological disposal. In this canister design, the vitrified nuclear waste stainless steel container is shielded by a 100 mm thick lead wall. For mechanical resistance, the canister will also have a thin carbon steel external liner. Experimental and mathematical modeling studies are aimed to asses the corrosion kinetics of the carbon steel liner in first instance and then, once this liner has been corroded away, the corrosion kinetics of the main lead barrier. Being that oxygen reduction is the main cathodic reaction that supports the anodic oxidation of iron, a model is described predicting the rate of oxygen consumption in a sealed deep nuclear waste disposal vault as a result of the canister corrosion. Oxidation processes other than container corrosion, and that can account also for oxygen depletion, are not taken into consideration. Corrosion experimental studies on lead and its alloys in groundwater are also reported. These experiments are aimed to improve the corrosion resistance of commercial lead in groundwater. (author)

Diversity of Metabolically Active Bacteria in Water-Flooded High-Temperature Heavy Oil Reservoir

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Tamara N. Nazina

2017-04-01

Full Text Available The goal of this work was to study the overall genomic diversity of microorganisms of the Dagang high-temperature oilfield (PRC and to characterize the metabolically active fraction of these populations. At this water-flooded oilfield, the microbial community of formation water from the near-bottom zone of an injection well where the most active microbial processes of oil degradation occur was investigated using molecular, cultural, radiotracer, and physicochemical techniques. The samples of microbial DNA and RNA from back-flushed water were used to obtain the clone libraries for the 16S rRNA gene and cDNA of 16S rRNA, respectively. The DNA-derived clone libraries were found to contain bacterial and archaeal 16S rRNA genes and the alkB genes encoding alkane monooxygenases similar to those encoded by alkB-geo1 and alkB-geo6 of geobacilli. The 16S rRNA genes of methanogens (Methanomethylovorans, Methanoculleus, Methanolinea, Methanothrix, and Methanocalculus were predominant in the DNA-derived library of Archaea cloned sequences; among the bacterial sequences, the 16S rRNA genes of members of the genus Geobacillus were the most numerous. The RNA-derived library contained only bacterial cDNA of the 16S rRNA sequences belonging to metabolically active aerobic organotrophic bacteria (Tepidimonas, Pseudomonas, Acinetobacter, as well as of denitrifying (Azoarcus, Tepidiphilus, Calditerrivibrio, fermenting (Bellilinea, iron-reducing (Geobacter, and sulfate- and sulfur-reducing bacteria (Desulfomicrobium, Desulfuromonas. The presence of the microorganisms of the main functional groups revealed by molecular techniques was confirmed by the results of cultural, radioisotope, and geochemical research. Functioning of the mesophilic and thermophilic branches was shown for the microbial food chain of the near-bottom zone of the injection well, which included the microorganisms of the carbon, sulfur, iron, and nitrogen cycles.

Apparent expression of flower colours and internal variation of enzyme activities in some typical phenotypes of dyer's saffron cultivars

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Koshi Saito

2014-01-01

Full Text Available Phytochemical screening of four Carthamus pigments in phenotypically different cultivars of dyer's saffron was carried out by means of chromatographic techniques. The pigment composition in the floral part correlated well with the flower colour, supporting these components as idoneous chemotaxonomic markers. Among seven cultivars examined, three were orange-yellow and contained carthamin (red and precarthamin, safflor yellow A and safflor yellow B (orange-yellow (type 0. There were bright-yellow and also had the above four pigments (type Y. The seventh cultivar was ivory-white and produced no quinoidal chalcones in the florets (type W. Relative activities of three different enzymes were examined in soluble protein extracts from etiolated seedlings of the garden varieties. Monophenol monooxygenase (EC 1.14.18.1 and peroxidase (EC 1.11.1.7. were distributed over all cultivars tested. The relative level of the enzyme activities could be ordered as follows: type 0, type W and type Y. The activity of a carthamin-synthesizing enzyme was found in the protein extracts from all garden forms examined. Its activity was most prominent in type O. The activity level in type W was inferior to that of type O. The catalytic intensity in type Y was found to even lower. The results were discussed as to the composition of the phenotypic markers and the distribution of the enzyme activities in three different garden forms of dyer's saffron cultivars.

Synthesis and Biological Activities of Novel Anthranilic Diamides Analogues Containing Benzo[b]thiophene

Institute of Scientific and Technical Information of China (English)

ZHANG Ji-feng; LIU Chen; LIU Peng-fei; YAN Tao; WANG Bao-lei; XIONG Li-xia; LI Zheng-ming

2013-01-01

A series of novel anthranilic diamides analogues containing benzo[b]thiophenyl ring was designed and synthesized.Their structures were characterized by melting points,1H nuclear magnetic resonance(1H NMR) and high-resolution mass spectrometry(HRMS).The bioassay tests indicate that their insecticidal activities were weak to moderate.Antibacterial tests indicate that some of the compounds showed favourable activity in vitro against Physalospora piricola,Alternaria solani,Cercospora arachidicola,Gibberella sanbinetti and Phytophthora infestans at a dosage of 50 mg/L.

Biological Sensors Using DNA Functionalized Multiwalled Carbon Nanotubes

Science.gov (United States)

2009-10-01

hydrodynamic voltammetry and the results have been discussed. 5 2. Experimental Methods Reagents GOD (EC 1.1.3.4, Aspergillus niger , >100 U...is of practical use, stable and inexpensive. GOD from Aspergillus , is a homodimer containing two tightly bound flavine adenine dinucleotide (FAD

Field experiment on spray drift: Deposition and airborne drift during application to a winter wheat crop

NARCIS (Netherlands)

Wolters, A.; Linnemann, V.; Zande, van de J.C.; Vereecken, H.

2008-01-01

A field experiment was performed to evaluate various techniques for measuring spray deposition and airborne drift during spray application to a winter wheat crop. The application of a spraying agent containing the fluorescent dye Brilliant Sulfo Flavine by a conventional boom sprayer was done

Antiparasitic Activity of Sulfur- and Fluorine-Containing Bisphosphonates against Trypanosomatids and Apicomplexan Parasites

Directory of Open Access Journals (Sweden)

Tamila Galaka

2017-01-01

Full Text Available Based on crystallographic data of the complexes 2-alkyl(aminoethyl-1,1-bisphosphonates–Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS and T gondii (TgFPPS, respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(aminoethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

Synthesis and Antifungal Activity of Novel Sulfone Derivatives Containing 1,3,4-Oxadiazole Moieties

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Maoguo Tong

2011-11-01

Full Text Available A series of new sulfone compounds containing 1,3,4-oxadiazole moieties were synthesized. The structures of these compounds were confirmed by spectroscopic data (IR, 1H- and 13C-NMR and elemental analyses. Antifungal tests indicated that all the title compounds exhibited good antifungal activities against eight kinds of plant pathogenic fungi, and some showed superiority over the commercial fungicide hymexazol. Among them, compounds 5d, 5e, 5f, and 5i showed prominent activity against B. cinerea, with determined EC50 values of 5.21 μg/mL, 8.25 µg/mL, 8.03 µg/mL, and 21.00 µg/mL, respectively. The present work demonstrates that sulfone derivatives such as 5d containing a 1,3,4-oxadiazole moiety can be used as possible lead compounds for the development of potential agrochemicals.

Pharmacological kynurenine 3-monooxygenase enzyme inhibition significantly reduces neuropathic pain in a rat model.

Science.gov (United States)

Rojewska, Ewelina; Piotrowska, Anna; Makuch, Wioletta; Przewlocka, Barbara; Mika, Joanna

2016-03-01

Recent studies have highlighted the involvement of the kynurenine pathway in the pathology of neurodegenerative diseases, but the role of this system in neuropathic pain requires further extensive research. Therefore, the aim of our study was to examine the role of kynurenine 3-monooxygenase (Kmo), an enzyme that is important in this pathway, in a rat model of neuropathy after chronic constriction injury (CCI) to the sciatic nerve. For the first time, we demonstrated that the injury-induced increase in the Kmo mRNA levels in the spinal cord and the dorsal root ganglia (DRG) was reduced by chronic administration of the microglial inhibitor minocycline and that this effect paralleled a decrease in the intensity of neuropathy. Further, minocycline administration alleviated the lipopolysaccharide (LPS)-induced upregulation of Kmo mRNA expression in microglial cell cultures. Moreover, we demonstrated that not only indirect inhibition of Kmo using minocycline but also direct inhibition using Kmo inhibitors (Ro61-6048 and JM6) decreased neuropathic pain intensity on the third and the seventh days after CCI. Chronic Ro61-6048 administration diminished the protein levels of IBA-1, IL-6, IL-1beta and NOS2 in the spinal cord and/or the DRG. Both Kmo inhibitors potentiated the analgesic properties of morphine. In summary, our data suggest that in neuropathic pain model, inhibiting Kmo function significantly reduces pain symptoms and enhances the effectiveness of morphine. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology and indicate that Kmo represents a novel pharmacological target for the treatment of neuropathy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Kinetic characterization of Vibrio cholerae ApbE: Substrate specificity and regulatory mechanisms

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Fang, Xuan; Liang, Pingdong; Raba, Daniel Alexander; Rosas-Lemus, Mónica; Chakravarthy, Srinivas; Tuz, Karina; Juárez, Oscar; Permyakov, Eugene A.

2017-10-24

ApbE is a member of a novel family of flavin transferases that incorporates flavin mononucleotide (FMN) to subunits of diverse respiratory complexes, which fulfill important homeostatic functions. In this work a detailed characterization of Vibrio cholerae ApbE physiologic activity, substrate specificity and pH dependency was carried out. The data obtained show novel characteristics of the regulation and function of this family. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, studies of the pH-dependency of the activity show that the reaction is favored under alkaline conditions, with a pKa of 8.4. These studies, together with sequence and structure analysis allowed us to identify His257, which is absolutely conserved in the family, as a candidate for the residue whose deprotonation controls the activity. Remarkably, the mutant H257G abolished the flavin transfer activity, strongly indicating that this residue plays an important role in the catalytic mechanism of ApbE.

From Plant Infectivity to Growth Patterns: The Role of Blue-Light Sensing in the Prokaryotic World

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Aba Losi

2014-01-01

Full Text Available Flavin-based photoreceptor proteins of the LOV (Light, Oxygen, and Voltage and BLUF (Blue Light sensing Using Flavins superfamilies are ubiquitous among the three life domains and are essential blue-light sensing systems, not only in plants and algae, but also in prokaryotes. Here we review their biological roles in the prokaryotic world and their evolution pathways. An unexpected large number of bacterial species possess flavin-based photosensors, amongst which are important human and plant pathogens. Still, few cases are reported where the activity of blue-light sensors could be correlated to infectivity and/or has been shown to be involved in the activation of specific genes, resulting in selective growth patterns. Metagenomics and bio-informatic analysis have only recently been initiated, but signatures are beginning to emerge that allow definition of a bona fide LOV or BLUF domain, aiming at better selection criteria for novel blue-light sensors. We also present here, for the first time, the phylogenetic tree for archaeal LOV domains that have reached a statistically significant number but have not at all been investigated thus far.

Activity of Fosfomycin- and Daptomycin-Containing Bone Cement on Selected Bacterial Species Being Associated with Orthopedic Infections

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Sigrun Eick

2017-01-01

Full Text Available The purpose of this study was to determine activity of fosfomycin/gentamicin and daptomycin/gentamicin-containing PMMA bone-cement against Staphylococcus aureus (MRSA, MSSA, Staphylococcus epidermidis, Enterococcus faecium (VRE, and E. coli (ESBL; only fosfomycin. Test specimens of the bone cement were formed and bacteria in two concentrations were added one time or repeatedly up to 96 h. All fosfomycin-containing cement killed ultimately all MSSA, Staphylococcus epidermidis, and E. coli within 24 h; growth of MRSA was suppressed up to 48 h. Activity of daptomycin-containing cement depended on the concentration; the highest concentrated bone cement used (1.5 g daptomycin/40 g of powder was active against all one-time added bacteria. When bacteria were added repeatedly to fosfomycin-containing cement, growth was suppressed up to 96 h and that of MRSA and VRE only up to 24 h. The highest concentration of daptomycin suppressed the growth of repeated added bacteria up to 48 h (VRE until 96 h (MSSA, MRSA. In conclusion, PMMA bone cement with 1.5 g of daptomycin and 0.5 g of gentamicin may be an alternative in treatment of periprosthetic infections caused by Gram-positive bacteria.

ORF Alignment: NC_004741 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004741 gi|30064861 >1ogiA 15 282 6 231 1e-18 ... ref|NP_709648.2| ferrisiderophore... reductase, flavin reductase (NADPH:flavin ... oxidoreductase) [Shigella flexneri 2a str. 301] ... gb|AAN45355.2| ferri...higella ... flexneri 2a str. 301] ref|NP_839032.1| ferrisiderophore ... ... ... gb|AAP18843.1| ferrisiderophore reductase, flavin ... reductase (NADPH:flavin oxidoreducta

ORF Alignment: NC_004337 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004337 gi|56480453 >1ogiA 15 282 6 231 1e-18 ... ref|NP_709648.2| ferrisiderophore... reductase, flavin reductase (NADPH:flavin ... oxidoreductase) [Shigella flexneri 2a str. 301] ... gb|AAN45355.2| ferri...higella ... flexneri 2a str. 301] ref|NP_839032.1| ferrisiderophore ... ... ... gb|AAP18843.1| ferrisiderophore reductase, flavin ... reductase (NADPH:flavin oxidoreducta

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

Science.gov (United States)

Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan; Dougherty, Vi

2017-02-14

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in k cat /K m and k red for (S)-6-hydroxynicotine, respectively, with larger effects on the k cat /K m value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the k cat /K m value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the k cat /K m value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

Bioanalytical methods for determination of tamoxifen and its phase I metabolites: A review

International Nuclear Information System (INIS)

Teunissen, S.F.; Rosing, H.; Schinkel, A.H.; Schellens, J.H.M.; Beijnen, J.H.

2010-01-01

The selective estrogen receptor modulator tamoxifen is used in the treatment of early and advanced breast cancer and in selected cases for breast cancer prevention in high-risk subjects. The cytochrome P450 enzyme system and flavin-containing monooxygenase are responsible for the extensive metabolism of tamoxifen into several phase I metabolites that vary in toxicity and potencies towards estrogen receptor (ER) alpha and ER beta. An extensive overview of publications on the determination of tamoxifen and its phase I metabolites in biological samples is presented. In these publications techniques were used such as capillary electrophoresis, liquid, gas and thin layer chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection, liquid scintillation counting and nuclear magnetic resonance spectroscopy). A trend is seen towards the use of liquid chromatography coupled to mass spectrometry (LC-MS). State-of-the-art LC-MS equipment allowed for identification of unknown metabolites and quantification of known metabolites reaching lower limit of quantification levels in the sub pg mL -1 range. Although tamoxifen is also metabolized into phase II metabolites, the number of publications reporting on phase II metabolism of tamoxifen is scarce. Therefore the focus of this review is on phase I metabolites of tamoxifen. We conclude that in the past decades tamoxifen metabolism has been studied extensively and numerous metabolites have been identified. Assays have been developed for both the identification and quantification of tamoxifen and its metabolites in an array of biological samples. This review can be used as a resource for method transfer and development of analytical methods used to support pharmacokinetic and pharmacodynamic studies of tamoxifen and its phase I metabolites.

On the problem of radiation purification of waste waters containing synthetic surface-active agents

International Nuclear Information System (INIS)

Buslaeva, S.P.; Kon'kov, N.G.; Makarochkina, L.M.; Panin, Yu.A.; Upadyshev, L.B.; Filippov, M.T.

1975-01-01

Radiation decomposition of artificial solutions containing surface-active substances and the real sewage of textile factories is studied. Experiments on treatment of sewage in a foamed state were conducted (since the irradiation of sewage in a liquid form did not result in the desired effect) with a laboratory installation that made it possible to irradiate sewage by an electron beam both under stationary and flowing conditions. The dose rate was about 140 w/kg. The results of determination of the dependence of the decomposition rate for solutions that contain surface-active substances on the absorbed dose and the magnitude of the beam current are presented. It is demonstrated that the decrease of dose rate is accompanied by the increase of the yield of the surface-active substance decomposition in the foam. During irradiation of real sewage the improvement of their coloring as well as the acceleration of coagulation and the sedimentation of dissolved and suspended substances were observed. Recommendations are suggested for design of an experimental-industrial installation for irradiation of sewage. It is established that with the efficiency of 1000 m 3 /day the cost of sewage treatment will be 35-40 kop/m 3

IN VITRO CHEMO-PREVENTATIVE ACTIVITY OF STRELITZIA NICOLAI ARIL EXTRACT CONTAINING BILIRUBIN.

Science.gov (United States)

Dwarka, Depika; Thaver, Veneesha; Naidu, Mickey; Koorbanally, Neil A; Baijnath, And Himansu

2017-01-01

The discovery of the only animal pigment, bilirubin, in the plant Strelitzia nicolai has triggered a vast number of questions regarding bilirubin's formation and its role in the human body. Recent studies have confirmed that bilirubin at certain levels have many medical benefits. Various case studies have revealed that bilirubin is a potent antioxidant. Cervical cancer is one of South Africa's largest womens' health crises. It is estimated that it affects one out of 41 South African women and kills approximately 8 women in the country every day. Thus, the aim of this study was to investigate if the aril extract of Strelitzia nicolai (Regel and Körn.) containing bilirubin possesses anti-cancer activity and to determine its effect on the induction of apoptosis. The DPPH activity was firstly used to determine the antioxidant effect of the extract. Thereafter, the cytotoxic effect was tested using the XTT assay. Apoptosis was confirmed and quantified using the Annexin V-PE kit and the morphology was studied using acridine orange and ethidium bromide. The aril extract decreased cell viability by 52% and induced apoptosis in HeLa cells; as shown by the Annexin V-PE Apoptosis detection kit and morphological studies with acridine orange/ethidium bromide staining. The activity of the extract as a potent antioxidant was immensely enhanced as compared to the bilirubin standard. These results suggest that S. nicolai aril extract containing bilirubin works synergistically as opposed to bilirubin on its own. Furthermore, this extract might be a good candidate for the therapeutic intervention of cervical cancer.

Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

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Antos, Piotr A; Błachuta, Małgorzata; Hrabia, Anna; Grzegorzewska, Agnieszka K; Sechman, Andrzej

2015-09-02

The aim of this in vitro study was to determine the effect of TCDD and luteinizing hormone (LH) on mRNA expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1), and the CYP1 family monooxygenases (CYP1A4, CYP1A5, CYP1B1), and to assess the basal and TCDD-induced activity of these enzymes in chicken ovarian follicles. White (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest preovulatory follicles (F3-F1) were exposed to TCDD (10nM), ovine LH (oLH; 10ng/mL) or a combination of TCDD (10nM) and oLH (10ng/mL), and increasing doses of TCDD (0.01-100nM). AHR1 and ARNT1 mRNA transcripts were found in all examined follicles. The effect of TCDD and oLH on AHR1 and ARNT1 mRNA expression depended on the maturational state of the follicle. CYP1A4 was predominantly expressed in the GL of the F3-F1 follicles; in comparison with the WF, a higher level of CYP1A5 mRNA was found both in the GL and TL of F3-F1 follicles. Alternatively, the highest level of CYP1B1 mRNA was noticed in the WF follicles. In different developmental stages of the follicle TCDD and oLH induced a different CYP1 isoform. TCDD increased EROD and MROD activities in all the investigated ovarian follicles. In conclusion, AHR1 and ARNT1 mRNA expression indicate that the chicken ovary is a target tissue for dioxin and dioxin-like compounds. The expression of CYP1-family genes and TCDD-inducible EROD and MROD activities in ovarian follicles suggest the possibility of xenobiotic detoxification in the chicken ovary. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Chemoenzymatic approaches to obtain chiral-centered selenium compounds

NARCIS (Netherlands)

Brondani, Patricia B.; Guilmoto, Nathalie M. A. F.; Dudek, Hanna M.; Fraaije, Marco W.; Andrade, Leandro H.

2012-01-01

The synthesis of chiral-centered selenium compounds is presented. Enantioselective oxidations of these organoselenium compounds were performed using a wide range of biocatalysts, including Baeyer-Villiger monooxygenases, oxidoreductases-containing Aspergillus terreus and lipase (Cal-B) in the

Oxidation of C18 Hydroxy-Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase-Related Hemoproteins Activated with Iodosylbenzene.

Science.gov (United States)

Teder, Tarvi; Boeglin, William E; Brash, Alan R

2017-07-01

Small catalase-related hemoproteins with a facility to react with fatty acid hydroperoxides were examined for their potential mono-oxygenase activity when activated using iodosylbenzene. The proteins tested were a Fusarium graminearum 41 kD catalase hemoprotein (Fg-cat, gene FGSG_02217), a Pseudomonas fluorescens Pfl01 catalase (37.5 kD, accession number WP_011333788.1), and a Mycobacterium avium ssp. paratuberculosis 33 kD catalase (gene MAP-2744c). 13-Hydroxy-octadecenoic acids (which are normally unreactive) were selected as substrates because these enzymes react specifically with the corresponding 13S-hydroperoxides (Pakhomova et al. 18:2559-2568, 5; Teder et al. 1862:706-715, 14). In the presence of iodosylbenzene Fg-cat converted 13S-hydroxy-fatty acids to two products: the 15,16-double bond of 13S-hydroxy α-linolenic acid was oxidized stereospecifically to the 15S,16R-cis-epoxide or the 13-hydroxyl was oxidized to the 13-ketone. Products were identified by UV, HPLC, LC-MS, NMR and by comparison with authentic standards prepared for this study. The Pfl01-cat displayed similar activity. MAP-2744c oxidized 13S-hydroxy-linoleic acid to the 13-ketone, and epoxidized the double bonds to form the 9,10-epoxy-13-hydroxy, 11,12-epoxy-13-hydroxy, and 9,10-epoxy-13-keto derivatives; equivalent transformations occurred with 9S-hydroxy-linoleic acid as substrate. In parallel incubations in the presence of iodosylbenzene, human catalase displayed no activity towards 13S-hydroxy-linoleic acid, as expected from the highly restricted access to its active site. The results indicated that with suitable transformation to Compound I, monooxygenase activity can be demonstrated by these catalase-related hemoproteins with tyrosine as the proximal heme ligand.

MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

Science.gov (United States)

Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance.

Science.gov (United States)

Liu, Simu; Bartnikas, Lisa M; Volko, Sigrid M; Ausubel, Frederick M; Tang, Dingzhong

2016-01-01

Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

Science.gov (United States)

Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

2015-11-01

The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. Copyright © 2015 Elsevier Inc. All rights reserved.

Mutation of the glucosinolate biosynthesis enzyme cytochrome P450 83A1 monooxygenase increases camalexin accumulation and powdery mildew resistance

Directory of Open Access Journals (Sweden)

Simu eLiu

2016-03-01

Full Text Available Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1, which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

Inhibiting prolyl isomerase activity by hybrid organic-inorganic molecules containing rhodium(II) fragments.

Science.gov (United States)

Coughlin, Jane M; Kundu, Rituparna; Cooper, Julian C; Ball, Zachary T

2014-11-15

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site. Copyright © 2014 Elsevier Ltd. All rights reserved.

Computation of the free energy change associated with one-electron reduction of coenzyme immersed in water: a novel approach within the framework of the quantum mechanical/molecular mechanical method combined with the theory of energy representation.

Science.gov (United States)

Takahashi, Hideaki; Ohno, Hajime; Kishi, Ryohei; Nakano, Masayoshi; Matubayasi, Nobuyuki

2008-11-28

The isoalloxazine ring (flavin ring) is a part of the coenzyme flavin adenine dinucleotide and acts as an active site in the oxidation of a substrate. We have computed the free energy change Deltamicro(red) associated with one-electron reduction of the flavin ring immersed in water by utilizing the quantum mechanical/molecular mechanical method combined with the theory of energy representation (QM/MM-ER method) recently developed. As a novel treatment in implementing the QM/MM-ER method, we have identified the excess charge to be attached on the flavin ring as a solute while the remaining molecules, i.e., flavin ring and surrounding water molecules, are treated as solvent species. Then, the reduction free energy can be decomposed into the contribution Deltamicro(red)(QM) due to the oxidant described quantum chemically and the free energy Deltamicro(red)(MM) due to the water molecules represented by a classical model. By the sum of these contributions, the total reduction free energy Deltamicro(red) has been given as -80.1 kcal/mol. To examine the accuracy and efficiency of this approach, we have also conducted the Deltamicro(red) calculation using the conventional scheme that Deltamicro(red) is constructed from the solvation free energies of the flavin rings at the oxidized and reduced states. The conventional scheme has been implemented with the QM/MM-ER method and the calculated Deltamicro(red) has been estimated as -81.0 kcal/mol, showing excellent agreement with the value given by the new approach. The present approach is efficient, in particular, to compute free energy change for the reaction occurring in a protein since it enables ones to circumvent the numerical problem brought about by subtracting the huge solvation free energies of the proteins in two states before and after the reduction.

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

Energy Technology Data Exchange (ETDEWEB)

Sukumar, Narayanasami [NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, Argonne, IL 60439 (United States); Dewanti, Asteriani [Department of Chemistry and Physics, Western Carolina University, Cullowhee, NC 28723 (United States); Merli, Angelo; Rossi, Gian Luigi [Department of Biochemistry and Molecular Biology, University of Parma, Parma (Italy); Mitra, Bharati [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Mathews, F. Scott, E-mail: mathews@biochem.wustl.edu [Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110 (United States); NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, Argonne, IL 60439 (United States)

2009-06-01

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

Synthesis and biological activity of the novel indanedione anticoagulant rodenticides containing fluorine

OpenAIRE

Chen, Feng; Liu, Liping; Bai, Zengguo; Zhang, Tianhua; Zhao, Keke

2016-01-01

Here, 3 fluorinated intermediates of drug were synthesized: (M1), (M2), (M3). Three new anticoagulant rodenticides were designed which were based on 4-hydroxycoumarin or 1,3-indandione, added acute toxicity groups containing fluorine. The structures of synthesized compounds were analyzed and proved by FT-IR spectroscopy and 1H nuclear magnetic resonance (1H-NMR). The compounds were also evaluated for their anticoagulant and acute biologic activity. In addition, both the acute orally toxicity ...

Catalytic activity of bimetal-containing Co,Pd systems in the oxidation of carbon monoxide

Science.gov (United States)

Oleksenko, L. P.; Lutsenko, L. V.

2013-02-01

The catalytic activity of low-percentage Co,Pd systems on ZSM-5, ERI, SiO2, and Al2O3 supports in the oxidation of CO was studied. The activity of bimetal-containing catalysts was shown to depend on the nature of the catalyst and the amount and ratio of their active components. According to the results of thermoprogrammed reduction with H2 (H2 TPR) and X-ray photoelectron spectroscopy (XPS) data, the metals are distributed as isolated cations or Coδ+-O-Pdδ+ clusters with cobalt and palladium cations surrounded by off-lattice oxygen in Co,Pd systems. The 0.8% Co,0.5% Pd-ZSM-5 bimetal catalysts were found to be more active due to the presence of clusters.

Two types of a passive safety containment for a near future BWR with active and passive safety systems

International Nuclear Information System (INIS)

Sato, Takashi; Akinaga, Makoto; Kojima, Yoshihiro

2009-01-01

The paper presents two types of a passive safety containment for a near future BWR. They are named Mark S and Mark X containment. One of their common merits is very low peak pressure at severe accidents without venting the containment atmosphere to the environment. The PCV pressure can be moderated within the design pressure. Another merit is the capability to submerge the PCV and the RPV above the core level. The third merit is robustness against external events such as a large commercial airplane crash. Both the containments have a passive cooling core catcher that has radial cooling channels. The Mark S containment is made of reinforced concrete and applicable to a large power BWR up to 1830 MWe. The Mark X containment has the steel secondary containment and can be cooled by natural circulation of outside air. It can accommodate a medium power BWR up to 1380 MWe. In both cases the plants have active and passive safety systems constituting in-depth hybrid safety (IDHS). The IDHS provides not only hardware diversity between active and passive safety systems but also more importantly diversity of the ultimate heat sinks between the atmosphere and the sea water. Although the plant concept discussed in the paper uses well-established technology, plant performance including economy is innovatively and evolutionally improved. Nothing is new in the hardware but everything is new in the performance.

Two types of a passive safety containment for a near future BWR with active and passive safety systems

Energy Technology Data Exchange (ETDEWEB)

Sato, Takashi [Toshiba Corporation, IEC, Gen-SS, 8, Shinsugita-ho, Isogo-ku, Yokohama (Japan)], E-mail: takashi44.sato@glb.toshiba.co.jp; Akinaga, Makoto; Kojima, Yoshihiro [Toshiba Corporation, IEC, Gen-SS, 8, Shinsugita-ho, Isogo-ku, Yokohama (Japan)

2009-09-15

The paper presents two types of a passive safety containment for a near future BWR. They are named Mark S and Mark X containment. One of their common merits is very low peak pressure at severe accidents without venting the containment atmosphere to the environment. The PCV pressure can be moderated within the design pressure. Another merit is the capability to submerge the PCV and the RPV above the core level. The third merit is robustness against external events such as a large commercial airplane crash. Both the containments have a passive cooling core catcher that has radial cooling channels. The Mark S containment is made of reinforced concrete and applicable to a large power BWR up to 1830 MWe. The Mark X containment has the steel secondary containment and can be cooled by natural circulation of outside air. It can accommodate a medium power BWR up to 1380 MWe. In both cases the plants have active and passive safety systems constituting in-depth hybrid safety (IDHS). The IDHS provides not only hardware diversity between active and passive safety systems but also more importantly diversity of the ultimate heat sinks between the atmosphere and the sea water. Although the plant concept discussed in the paper uses well-established technology, plant performance including economy is innovatively and evolutionally improved. Nothing is new in the hardware but everything is new in the performance.

Elaboration of Copper-Oxygen Mediated C–H Activation Chemistry in Consideration of Future Fuel and Feedstock Generation

Science.gov (United States)

Lee, Jung Yoon; Karlin, Kenneth D

2015-01-01

To contribute solutions for current energy concerns, improvements in the efficiency of C-H bond cleavage chemistry, e.g., selective oxidation of methane to methanol, could minimize losses in natural gas usage or produce feedstocks for fuels. Oxidative C-H activation is also a component of polysaccharide degradation, affording alternative biofuels from abundant biomass. Thus, an understanding of active-site chemistry in copper monooxygenases, those activating strong C-H bonds is briefly reviewed. Then, recent advances in the synthesis-generation and study of various copper-oxygen intermediates are highlighted. Of special interest are cupric-superoxide, Cu-hydroperoxo and Cu-oxy complexes. Such investigations can contribute to an enhanced future application of C-H oxidation or oxygenation processes using air, as concerning societal energy goals. PMID:25756327

Investigation of the enzyme system of detoxification of insecticides in the Colorado beetle

International Nuclear Information System (INIS)

Leonova, I.N.; Nedel'kina, S.V.; Salganik, R.I.

1986-01-01

The activity of three enzymes systems of xenobiotic metabolism - cytochrome P-450-dependent monooxygenases, nonspecific esterases, and glutathione S-transferases - was investigated at various stages of the development of the Colorado beetle Leptinotarsa decemlineata. Substantial sex and ontogenetic differences in the content of cytochrome P-450, the position of the maxima of the CO-differential spectra of its reduced form, and the substrate specificity of cytochrome P-450 were demonstrated. An increase in the activity of nonspecific esterases with increasing age of Colorado beetle larvae was observed. The insecticide 1-naphtholenol methylcarbamate, which is metabolized by the system of cytochrome P-450-dependent monooxygenases, is more toxic at the larval stage of development in comparison with the imaginal stage, which is in good agreement with the activity of this system at different stages of development. The inhibitor of microsomal monooxygenases piperonyl butoxide more than doubles the toxicity of the insecticide in the Colorado beetle imago. The data presented are evidence of a different contribution of the systems of detoxification to the sensitivity of the Colorado beetle to insecticides at different stages of metamorphosis

Free-Standing Metal Oxide Nanoparticle Superlattices Constructed with Engineered Protein Containers Show in Crystallo Catalytic Activity.

Science.gov (United States)

Lach, Marcel; Künzle, Matthias; Beck, Tobias

2017-12-11

The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

International Nuclear Information System (INIS)

Liu Jingquan; Paddon-Row, Michael N.; Gooding, J. Justin

2006-01-01

An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase - a probable consequence of the FAD being immersed in a protein environment - was responsible for the inability of the enzyme to be turned over under anaerobic conditions

Structural analysis and visible light-activated photocatalytic activity of iron-containing soda lime aluminosilicate glass

Energy Technology Data Exchange (ETDEWEB)

Iida, Yusuke; Akiyama, Kazuhiko [Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachi-Oji, Tokyo 192-0397 (Japan); Kobzi, Balázs; Sinkó, Katalin; Homonnay, Zoltán [Institute of Chemistry, Eötvös Loránd University, Pázmany P. s., 1/A, Budapest 1117 (Hungary); Kuzmann, Ernő [Institute of Chemistry, Eötvös Loránd University, Pázmany P. s., 1/A, Budapest 1117 (Hungary); Laboratory of Nuclear Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest 1512 (Hungary); Ristić, Mira; Krehula, Stjepko [Division of Materials Chemistry, RuđerBošković Institute, Bijenička cesta 54, Zagreb 10000 (Croatia); Nishida, Tetsuaki [Department of Biological and Environmental Chemistry, Faculty of Humanity-Oriented Science and Engineering, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555 (Japan); Kubuki, Shiro, E-mail: kubuki@tmu.ac.jp [Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachi-Oji, Tokyo 192-0397 (Japan)

2015-10-05

Highlights: • Hematite was precipitated by heat treatment of iron aluminosilicate glass. • The hematite phase shows visible light photocatalytic activity. • We could prepare an effective photocatalyst from ‘ubiquitous elements’. - Abstract: A relationship between structure and visible light-activated photocatalytic activity of iron-containing soda lime aluminosilicate (15Na{sub 2}O⋅15CaO⋅40Fe{sub 2}O{sub 3}⋅xAl{sub 2}O{sub 3}⋅(30−x)SiO{sub 2}) glass (xNCFAS) was investigated by means of {sup 57}Fe-Mössbauer spectroscopy, X-ray diffractometry (XRD) and UV–visible light absorption spectroscopy (UV–VIS). The {sup 57}Fe-Mössbauer spectrum of 11NCFAS glass measured after heat-treatment at 1000 °C for 100 min was composed of a paramagnetic doublet due to Fe{sup III}(T{sub d}) and two magnetic sextets due to regular hematite (α-Fe{sub 2}O{sub 3}) and hematite with larger internal magnetic field. X-ray diffraction patterns of heat-treated xNCFAS samples resulted in decrease of α-Fe{sub 2}O{sub 3} and increase of Ca{sub 2}Fe{sub 22}O{sub 33} or CaFe{sub 2}O{sub 4} with alumina content. A quick decrease in methylene blue (MB) concentration from 15.6 to 4.7 μmol L{sup −1} was observed in the photocatalytic reaction test with 40 mg of heat-treated 11NCFAS glass under visible light-exposure. The largest first-order rate constant of MB decomposition (k) was estimated to be 9.26 × 10{sup −3} min{sup −1}. Tauc’s plot yielded a band gap energy (E{sub g}) of 1.88 eV for heat-treated 11NCFAS glass, which is smaller than previously reported E{sub g} of 2.2 eV for α-Fe{sub 2}O{sub 3}. These results prove that addition of Al{sub 2}O{sub 3} into iron-containing soda lime silicate glass is favorable for the preparation of improved visible light-photocatalyst with ‘ubiquitous’ elements.

HepG2 cells develop signs of riboflavin deficiency within four days of culture in riboflavin-deficient medium*

OpenAIRE

Werner, Ricarda; Manthey, Karoline C.; Griffin, Jacob B.; Zempleni, Janos

2005-01-01

Flavin mononucleotide and flavin adenine dinucleotide are essential coenzymes in redox reactions. For example, flavin adenine dinucleotide is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/L) medium for up to six days; controls ...

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing.

Science.gov (United States)

Radajewski, Stefan; Webster, Gordon; Reay, David S; Morris, Samantha A; Ineson, Philip; Nedwell, David B; Prosser, James I; Murrell, J Colin

2002-08-01

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further

Sequencing and characterization of mixed function monooxygenase genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like compounds

International Nuclear Information System (INIS)

Zhang Xiaowei; Moore, Jeremy N.; Newsted, John L.; Hecker, Markus; Zwiernik, Matthew J.; Jones, Paul D.; Bursian, Steven J.

2009-01-01

As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and CYP1A2

Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes.

Science.gov (United States)

Steinhagen, Folkert; Zillinger, Thomas; Peukert, Konrad; Fox, Mario; Thudium, Marcus; Barchet, Winfried; Putensen, Christian; Klinman, Dennis; Latz, Eicke; Bode, Christian

2018-04-01

Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi-Goutières Syndrome (AGS). The recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1-deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self-DNA. Collectively, these findings might lead to the development of new therapeutics against IFN-driven pathologies due to cGAS activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrochemical Characterization of Escherichia coli Adaptive Response Protein AidB

Directory of Open Access Journals (Sweden)

Sean J. Elliott

2012-12-01

Full Text Available When exposed to known DNA-damaging alkylating agents, Escherichia coli cells increase production of four DNA repair enzymes: Ada, AlkA, AlkB, and AidB. The role of three enzymes (Ada, AlkA, and AlkB in repairing DNA lesions has been well characterized, while the function of AidB is poorly understood. AidB has a distinct cofactor that is potentially related to the elusive role of AidB in adaptive response: a redox active flavin adenine dinucleotide (FAD. In this study, we report the thermodynamic redox properties of the AidB flavin for the first time, both for free protein and in the presence of potential substrates. We find that the midpoint reduction potential of the AidB flavin is within a biologically relevant window for redox chemistry at −181 mV, that AidB significantly stabilizes the flavin semiquinone, and that small molecule binding perturbs the observed reduction potential. Our electrochemical results combined with structural analysis allow for fresh comparisons between AidB and the homologous acyl-coenzyme A dehydrogenase (ACAD family of enzymes. AidB exhibits several discrepancies from ACADs that suggest a novel catalytic mechanism distinct from that of the ACAD family enzymes.

Potential anticancer activity of curcumin analogs containing sulfone on human cancer cells

Directory of Open Access Journals (Sweden)

Zhang Qiuyan

2016-01-01

Full Text Available Three curcumin analogs(S1-S3 containing sulfone were investigated for their effects on human prostate cancer PC-3, colon cancer HT-29, lung cancer H1299 and pancreatic cancer BxPC-3 cells. The three compounds were approximately 16-to 96-fold more active than curcumin in these cell lines as determined by the MTT assay. The effects of these compounds on cell growth were further studied in prostate cancer PC-3 cells in both two dimensional (2D and three dimensional (3D cultures. S1-S3strongly inhibited the growth and induced cell death in PC-3 cells, and the effects of these compounds were associated with suppression of nuclear factor kappa B (NF-κB transcriptional activity. Moreover, treatment of PC-3 cells with all three compounds caused a decrease in the level of phosphorylated signal transducer and activator of transcription-3 (p-STAT3 (Tyr705,but not p-STAT3(Ser727. Only S1and S2decreased the presence of phosphorylated Akt (p-Akt in PC-3 cells. These curcumin analogs warrant further in vivo studies for anticancer activities in suitable animal models.

Hydroxylamine addition impact to Nitrosomonas europaea activity in the presence of monochloramine

Science.gov (United States)

In drinking water, monochloramine may promote ammonia–oxidizing bacteria (AOB) growth because of concurrent ammonia presence. AOB use (i) ammonia monooxygenase for biological ammonia oxidation to hydroxylamine and (ii) hydroxylamine oxidoreductase for hydroxylamine oxidation to ...

Influence of metal coating methods on the activity of bimetal-containing zeolite catalysts of Co, Pd-ZSM-5 in carbon monoxide oxidation

Science.gov (United States)

Oleksenko, L. P.; Lutsenko, L. V.; Yatsimirskii, V. K.

2011-07-01

It has been established that catalytic activity in the CO oxidation of bimetal-containing zeolite Co,Pd-systems based on ZSM-5 and obtained via ion exchange and impregnation at different orders of the introduction of metal cations is higher than that of monometal-containing systems Co-ZSM-5 and Pd—ZSM-5. Through TPD of NH3, it was determined that coordination-unsaturated bicationic associates are formed in Co,Pd/ZSM-5 zeolites obtained by ion exchange. It was found that the activity of bimetal-containing systems depends on the relation of the active components.

[Altered expressions of alkane monooxygenase and hypoxia inducible factor-1α expression in lung tissue of rat hypoxic pulmonary hypertension].

Science.gov (United States)

Deng, Hua-jun; Yuan, Ya-dong

2013-10-29

To explore the altered expressions of alkane monooxygenase (AlkB) and hypoxia-inducible factor-1α (HIF-1α) in a rat model of hypoxic pulmonary arterial hypertension. Twenty Wistar rats were divided randomly into normal control and hypoxia groups after 1-week adaptive feeding. Hypoxia group was raised in a homemade organic glass tank with a 24-h continuous supply of air and nitrogen atmospheric mixed gas. And the oxygen concentration of (10.0 ± 0.5)% was controlled by oxygen monitoring control system. The control group was maintained in room air. Both groups stayed in the same room with the same diet. After 8 weeks, the level of mean pulmonary pressure (mPAP) was measured by right-heart catheterization, right ventricular hypertrophy index (RVHI) calculated by the ratio of right ventricle to left ventricle plus septum and hypoxic pulmonary vascular remodeling (HPSR) observed under microscope. And the levels of AlkB and HIF-1α mRNA and protein in lungs were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. At 8 weeks post-hypoxia, compared with the control group [11.0 ± 0.7 mm Hg (1 mm Hg = 0.133 kPa), 0.210 ± 0.035], the levels of mPAP and RVHI in hypoxia group (33.3 ± 1.3 mm Hg, 0.448 ± 0.013) increased significantly (both P < 0.05), the expressions of AlkB mRNA and protein in pulmonary tissue decreased significantly (0.338 ± 0.085 vs 0.688 ± 0.020, P < 0.01) (0.483 ± 0.052 vs 0.204 ± 0.010, P < 0.01), and the expressions of HIF-1α mRNA and protein increased significantly (0.790 ± 0.161 vs 0.422 ± 0.096, P < 0.01) (0.893 ± 0.080 vs 0.346 ± 0.008, P < 0.01). The down-regulation of AlkB in lung tissue may increase the activity of HIF-1 to participate in the occurrence and development of pulmonary hypertension.

Factor analysis in optimization of formulation of high content uniformity tablets containing low dose active substance.

Science.gov (United States)

Lukášová, Ivana; Muselík, Jan; Franc, Aleš; Goněc, Roman; Mika, Filip; Vetchý, David

2017-11-15

Warfarin is intensively discussed drug with narrow therapeutic range. There have been cases of bleeding attributed to varying content or altered quality of the active substance. Factor analysis is useful for finding suitable technological parameters leading to high content uniformity of tablets containing low amount of active substance. The composition of tabletting blend and technological procedure were set with respect to factor analysis of previously published results. The correctness of set parameters was checked by manufacturing and evaluation of tablets containing 1-10mg of warfarin sodium. The robustness of suggested technology was checked by using "worst case scenario" and statistical evaluation of European Pharmacopoeia (EP) content uniformity limits with respect to Bergum division and process capability index (Cpk). To evaluate the quality of active substance and tablets, dissolution method was developed (water; EP apparatus II; 25rpm), allowing for statistical comparison of dissolution profiles. Obtained results prove the suitability of factor analysis to optimize the composition with respect to batches manufactured previously and thus the use of metaanalysis under industrial conditions is feasible. Copyright © 2017 Elsevier B.V. All rights reserved.

Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions

International Nuclear Information System (INIS)

Estabrook, Ronald W.

2005-01-01

The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11β-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O 18 studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17α-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17α-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11β-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction

Multiple containment for LSA [low specific activity] and SCO [surface contaminated objects] wastes

International Nuclear Information System (INIS)

Burgess, M.H.

1993-09-01

Radioactive wastes are generally transported in the form of Low Specific Activity (LSA) materials or Surface Contaminated Objects (SCO). This report proposes that a method of acknowledging the beneficial effects of multiple containment for such wastes should be written into the 1996 Edition of the IAEA Transport Regulations. Experience used to assess risks from on-site movements of radioactive material in the UK can be applied to develop safety arguments justifying the alleviation of off-site transport risks. (UK)

Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment.

Directory of Open Access Journals (Sweden)

Joshua B Parsons

2010-11-01

Full Text Available PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(IIIalamin to cob(IIalamin and cob(IIalamin to cob(Ialamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

Peripheral kynurenine-3-monooxygenase deficiency as a potential risk factor for metabolic syndrome in schizophrenia patients.

Science.gov (United States)

Oxenkrug, Gregory; van der Hart, Marieke; Roeser, Julien; Summergrad, Paul

2017-01-01

Increased predisposition of schizophrenia patients (SP) to development of obesity and insulin resistance suggested common signaling pathway between metabolic syndrome (MetS) and schizophrenia. Deficiency of kynurenine-3-monooxygenase (KMO), enzyme catalyzing formation of 3-hydroxykynurenine (3-HK) from kynurenine (Kyn), a tryptophan (Trp) metabolite, might contribute to development of MetS as suggested by non-expression of KMO genes in human fat tissue and elevated serum concentrations of Kyn and its metabolites, kynurenic (KYNA) and anthranilic (ANA) acids, in diabetic patients and Zucker fatty rats (ZFR). Markers of KMO deficiency: decreased 3-HK and elevated Kyn, KYNA and ANA, were observed in brains and spinal fluids of SP, and in brains and serum of experimental animals with genetically- or pharmacologically-induced KMO deficiency. However, elevated concentrations of ANA and decreased 3-HK were reported in serum of SP without concurrent increase of Kyn and KYNA. Present study aimed to re-assess serum Kyn metabolites (HPLC-MS) in a sub-group of SP with elevated KYNA. We found increased Kyn concentrations (by 30%) and Kyn:Trp ratio (by 20%) in serum of SP with elevated KYNA concentrations (by 40%). Obtained results and our previous data suggest that peripheral KMO deficiency might be manifested by, at least, two different patterns: elevated ANA with decreased 3-HK; and elevated KYNA and KYN. The latter pattern was previously described in type 2 diabetes patients and might underline increased predisposition of SP to development of MetS. Assessment of peripheral KMO deficiency might identify SP predisposed to MetS. Attenuation of the consequences of peripheral KMO deficiency might be a new target for prevention/treatment of obesity and diabetes in SP.

Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases