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Sample records for multicomponent monooxygenase pseudomonas

  1. Substrate Trafficking And Dioxygen Activation in Bacterial Multicomponent Monooxygenases

    SciTech Connect

    Murray, L.J.; Lippard, S.J.

    2009-06-03

    Non-heme carboxylate-bridged diiron centers in the hydroxylase components of the bacterial multicomponent monooxygenases process four substrates during catalysis: electrons, protons, dioxygen, and hydrocarbons. Understanding how protein-protein interactions mediate the transport of these substrates to the diiron center to achieve the selective oxidation of the hydrocarbon is a significant challenge. In this Account, we summarize our current knowledge of these processes with a focus on the methane monooxygenase system. We also describe recent results for the toluene/o-xylene monooxygenase and phenol hydroxylase systems from Pseudomonas sporium OX1. The observation in these latter systems of a diiron(III) oxygenated intermediate having different Moessbauer parameters from analogous species in other carboxylate-bridged diiron proteins is discussed. The results indicate that the ability of the protein framework to tune the reactivity of the diiron center at structurally similar active sites is substantially more complex than previously recognized.

  2. Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1

    SciTech Connect

    Bertoni, G.; Martino, M.; Galli, E.; Barbieri, P.

    1998-10-01

    The toluene/o-xylene monooxygenase cloned from Pseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from Pseudomonas strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase in Escherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

  3. Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from pseudomonas sp. strain JS150

    SciTech Connect

    Johnson, G.R.; Olsen, R.H.

    1995-09-01

    Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, strain JS150 was also found to carry genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen ({sup 18}O{sub 2}) incorporation experiments using Pseudomonas aeruginosa strains carrying the cloned genes confirmed toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Encoding the toluene-2-monooxygenase and regulatory gene product was localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined, revealing six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. All the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomo-KR1 and Burkholderia (Pseudomonas) picketti PKO1. The relationship found between the phenol hydroxlases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest. 58 refs., 8 figs., 1 tab.

  4. Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.

    PubMed

    Wang, Weixue; Liang, Alexandria D; Lippard, Stephen J

    2015-09-15

    A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the

  5. Molecular Determinants of the Regioselectivity of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1▿ †

    PubMed Central

    Notomista, Eugenio; Cafaro, Valeria; Bozza, Giuseppe; Di Donato, Alberto

    2009-01-01

    Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori. PMID:19074607

  6. Molecular determinants of the regioselectivity of toluene/o-xylene monooxygenase from Pseudomonas sp. strain OX1.

    PubMed

    Notomista, Eugenio; Cafaro, Valeria; Bozza, Giuseppe; Di Donato, Alberto

    2009-02-01

    Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori. PMID:19074607

  7. Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase

    SciTech Connect

    Kwangmu Yen; Karl, M.R.; Blatt, L.M.; Simon, M.J.; Winter, R.B.; Fausset, P.R.; Lu, H.S.; Harcourt, A.A.; Chen, K.K. )

    1991-09-01

    Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The authors have cloned and characterized a gene cluster from KR 1 that determines the T4MO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB 101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but no on toluene. An insert consisting of two SacI fragments of identical size was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport of the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.

  8. Enhancing Indigo Production by Over-Expression of the Styrene Monooxygenase in Pseudomonas putida.

    PubMed

    Cheng, Lei; Yin, Sheng; Chen, Min; Sun, Baoguo; Hao, Shuai; Wang, Chengtao

    2016-08-01

    As an important traditional blue dye, indigo has been used in food and textile industry for centuries, which can be produced via the styrene oxygenation pathway in Pseudomonas putida. Hence, the styrene monooxygenase gene styAB and oxide isomerase gene styC are over-expressed in P. putida to investigate their roles in indigo biosynthesis. RT-qPCR analysis indicated that transcriptions of styA and styB were increased by 2500- and 750-folds in the styAB over-expressed strain B4-01, compared with the wild-type strain B4, consequently significantly enhancing the indole monooxygenase activity. Transcription of styC was also increased by 100-folds in the styC over-expressed strain B4-02. Besides, styAB over-expression slightly up-regulated the transcription of styC in B4-01, while styC over-expression hardly exerted an effect on the transcriptional levels of styA and styB and indole monooxygenase activity in B4-02. Furthermore, shaking flask experiments showed that indigo production in B4-01 reached 52.13 mg L(-1) after 24 h, which was sevenfold higher than that in B4. But no obvious increase in indigo yield was observed in B4-02. Over-expression of styAB significantly enhanced the indigo production, revealing that the monooxygenase STYAB rather than oxide isomerase STYC probably acted as the key rate-limiting enzyme in the indigo biosynthesis pathway in P. putida. This work provided a new strategy for enhancing indigo production in Pseudomonas. PMID:27154464

  9. A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from Pseudomonas fluorescens ST

    PubMed Central

    Di Gennaro, Patrizia; Colmegna, Andrea; Galli, Enrica; Sello, Guido; Pelizzoni, Francesca; Bestetti, Giuseppina

    1999-01-01

    We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals. PMID:10347083

  10. 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

    SciTech Connect

    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.

    2015-10-31

    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.

  11. 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

    PubMed Central

    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 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. PMID:26527149

  12. Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

    PubMed Central

    Yee, Dennis C.; Maynard, Jennifer A.; Wood, Thomas K.

    1998-01-01

    Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA+ (toluene o-monooxygenase) genes from Burkholderia cepacia PR123(TOM23C). A transposon integration vector was used to insert tomA+ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR123(TOM23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h−1) and colonized wheat (3 × 106 CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h−1; level of colonization, 4 × 106 CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil. PMID:9435067

  13. Oxidation of trichloroethylene, 1,1-dichloroethylene, and chloroform by toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1

    SciTech Connect

    Chauhan, S.; Wood, T.K.; Barbieri, P.

    1998-08-01

    Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE, 1,1-DCE, and chloroform at initial rates of 3.1, 3.6, and 1.6 nmol, respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization. Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.

  14. Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism.

    PubMed

    Meneely, Kathleen M; Lamb, Audrey L

    2007-10-23

    Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. PMID:17900176

  15. Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352

    PubMed Central

    Bhushan, Bharat; Halasz, Annamaria; Spain, Jim C.; Hawari, Jalal

    2004-01-01

    CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C6H6N12O12), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min−1 mg of protein−1 under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M − H]− at 345 Da, corresponding to an empirical formula of C6H6N10O8, produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M − H]− at 381 Da corresponding to an empirical formula of C6H10N10O10. The latter was a hydrated product of the metabolite C6H6N10O8 with addition of two H2O molecules, as confirmed by tests using 18O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical. PMID:15240281

  16. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    SciTech Connect

    Gohain, Neelakshi; Thomashow, Linda S.; Mavrodi, Dmitri V.; Blankenfeldt, Wulf

    2006-10-01

    PhzS, an FAD-dependent monooxygenase that catalyzes a reaction involved in the biosynthesis of the virulence factor pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and seleno-l-methionine-labelled crystals is reported. The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 Å, α = γ = 90, β = 110.5°. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 Å. Seleno-l-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 Å, α = γ = 90, β = 110.5° and the diffraction limit is 2.7 Å.

  17. Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3▿

    PubMed Central

    Zhang, Jun-Jie; Liu, Hong; Xiao, Yi; Zhang, Xian-En; Zhou, Ning-Yi

    2009-01-01

    Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked. PMID:19218392

  18. Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16.

    PubMed

    Yu, Hao; Hausinger, Robert P; Tang, Hong-Zhi; Xu, Ping

    2014-10-17

    6-Hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB), a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, catalyzes pyridine-ring β-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine. Here, we generated His6-tagged HspB in Escherichia coli, characterized the properties of the recombinant enzyme, and investigated its mechanism of catalysis. In contrast to conclusions reported previously, the second product of the HspB reaction was shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H2O. Phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB. The reduction of HspB (with maxima at 375 and 460 nm, and a shoulder at 485 nm) by NADH was followed by stopped-flow spectroscopy, and the rate constant for reduction was shown to be stimulated by HSP. Reduced HspB reacts with oxygen to form a C(4a)-(hydro)peroxyflavin intermediate with an absorbance maximum at ∼400 nm within the first few milliseconds before converting to the oxidized flavoenzyme species. The formed C(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that hydrolyzes to the products 2,5-dihydroxypyridine and succinate. The investigation on the catalytic mechanism of a flavoprotein pyridine-ring β-position hydroxylase provides useful information for the biosynthesis of pyridine derivatives. PMID:25172510

  19. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE. PMID:25432418

  20. OXIDATION OF BIPHENYL BY A MULTICOMPONENT ENZYME SYSTEM FROM PSEUDOMONAS SP. STRAIN LB400

    EPA Science Inventory

    Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of bipheny...

  1. OXIDATION OF BIPHENYL BY A MULTICOMPONENT ENZYME SYSTEM FROM PSEUDOMONAS SP. STRAIN LB400

    EPA Science Inventory

    Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. his organism also cooxidizes several chlorinated biphenyl congeners. iphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl ...

  2. A Flexible Glutamine Regulates the Catalytic Activity of Toluene o-Xylene Monooxygenase

    PubMed Central

    2015-01-01

    Toluene/o-xylene monooxygenase (ToMO) is a bacterial multicomponent monooxygenase capable of oxidizing aromatic substrates. The carboxylate-rich diiron active site is located in the hydroxylase component of ToMO (ToMOH), buried 12 Å from the surface of the protein. A small, hydrophilic pore is the shortest pathway between the diiron active site and the protein exterior. In this study of ToMOH from Pseudomonas sp. OX1, the functions of two residues lining this pore, N202 and Q228, were investigated using site-directed mutagenesis. Steady-state characterization of WT and the three mutant enzymes demonstrates that residues N202 and Q228 are critical for turnover. Kinetic isotope effects and pH profiles reveal that these residues govern the kinetics of water egress and prevent quenching of activated oxygen intermediates formed at the diiron active site. We propose that this activity arises from movement of these residues, opening and closing the pore during catalysis, as seen in previous X-ray crystallographic studies. In addition, N202 and Q228 are important for the interactions of the reductase and regulatory components to ToMOH, suggesting that they bind competitively to the hydroxylase. The role of the pore in the hydroxylase components of other bacterial multicomponent monooxygenases within the superfamily is discussed in light of these conclusions. PMID:24873259

  3. The Combined Structural and Kinetic Characterization of a Bacterial Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes NMO Class I and II*

    PubMed Central

    Salvi, Francesca; Agniswamy, Johnson; Yuan, Hongling; Vercammen, Ken; Pelicaen, Rudy; Cornelis, Pierre; Spain, Jim C.; Weber, Irene T.; Gadda, Giovanni

    2014-01-01

    Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBankTM as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with kcatapp/Kmapp of 12 × 106 m−1 s−1, kcatapp of 1300 s−1, and Kmapp of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO. PMID:25002579

  4. Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453.

    PubMed

    Leisch, Hannes; Shi, Rong; Grosse, Stephan; Morley, Krista; Bergeron, Hélène; Cygler, Miroslaw; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C K

    2012-04-01

    A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140-152, 1983). Here we cloned and overexpressed the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP(+) at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP(+). A comparison of several crystal forms of OTEMO bound to FAD and NADP(+) revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k(cat)/K(m)) favors 2-n-hexyl cyclopentanone (4.3 × 10(5) M(-1) s(-1)) as a substrate, although its affinity (K(m) = 32 μM) was lower than that of the CoA-activated substrate (K(m) = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members. PMID:22267661

  5. Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

    PubMed Central

    Leisch, Hannes; Shi, Rong; Grosse, Stephan; Morley, Krista; Bergeron, Hélène; Cygler, Miroslaw; Iwaki, Hiroaki; Hasegawa, Yoshie

    2012-01-01

    A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1 s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (Km = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members. PMID:22267661

  6. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, tha...

  7. Camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions.

    PubMed

    Iwaki, Hiroaki; Grosse, Stephan; Bergeron, Hélène; Leisch, Hannes; Morley, Krista; Hasegawa, Yoshie; Lau, Peter C K

    2013-05-01

    Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 μM; kcat = 283 s(-1)) in preference to flavin adenine dinucleotide (FAD) (Km = 19 μM; kcat = 128 s(-1)). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates. PMID:23524667

  8. Membrane-association determinants of the omega-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa.

    PubMed

    Imperi, Francesco; Putignani, Lorenza; Tiburzi, Federica; Ambrosi, Cecilia; Cipollone, Rita; Ascenzi, Paolo; Visca, Paolo

    2008-09-01

    The L-ornithine N(delta)-oxygenase PvdA catalyses the N(delta)-hydroxylation of L-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa DeltapvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain. PMID:18757814

  9. The role of substrate binding pocket residues phenylalanine 176 and phenylalanine 196 on Pseudomonas sp. OX1 toluene o-xylene monooxygenase activity and regiospecificity.

    PubMed

    Sönmez, Burcu; Yanık-Yıldırım, K Cansu; Wood, Thomas K; Vardar-Schara, Gönül

    2014-08-01

    Saturation mutagenesis was used to generate eleven substitutions of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions F176 and F196 among which nine were novel: F176H, F176N, F176S, F176T, F196A, F196L, F196T, F196Y, F196H, F196I, and F196V. By testing the substrates phenol, toluene, and naphthalene, these positions were found to influence ToMO oxidation activity and regiospecificity. Specifically, TouA variant F176H was identified that had 4.7-, 4.3-, and 1.8-fold faster hydroxylation activity towards phenol, toluene, and naphthalene, respectively, compared to native ToMO. The F176H variant also produced the novel product hydroquinone (61%) from phenol, made twofold more 2-naphthol from naphthalene (34% vs. 16% by the wild-type ToMO), and had the regiospecificity of toluene changed from 51% to 73% p-cresol. The TouA F176N variant had the most para-hydroxylation capability, forming p-cresol (92%) from toluene and hydroquinone (82%) from phenol as the major product, whereas native ToMO formed 30% o-cresol, 19% m-cresol, and 51% of p-cresol from toluene and 100% catechol from phenol. For naphthalene oxidation, TouA variant F176S exhibited the largest shift in the product distribution by producing threefold more 2-naphthol. Among the other F196 variants, F196L produced catechol from phenol two times faster than the wild-type enzyme. The TouA F196I variant produced twofold less o-cresol and 19% more p-cresol from toluene, and the TouA F196A variant produced 62% more 2-naphthol from naphthalene compared to wild-type ToMO. Both of these positions have never been studied through the saturation mutagenesis and some of the best substitutions uncovered here have never been predicted and characterized for aromatics hydroxylation. PMID:24519264

  10. The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa

    PubMed Central

    Redelberger, David; Fadel, Firas; Filloux, Alain; Sivaneson, Melissa; de Bentzmann, Sophie; Bordi, Christophe

    2016-01-01

    In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response. PMID:27176226

  11. Functional redundancy in phenol and toluene degradation in Pseudomonas stutzeri strains isolated from the Baltic Sea.

    PubMed

    Heinaru, Eeva; Naanuri, Eve; Grünbach, Maarja; Jõesaar, Merike; Heinaru, Ain

    2016-09-01

    In the present study we describe functional redundancy of bacterial multicomponent monooxygenases (toluene monooxygenase (TMO) and toluene/xylene monooxygenase (XylAM) of TOL pathway) and cooperative genetic regulation at the expression of the respective catabolic operons by touR and xylR encoded regulatory circuits in five phenol- and toluene-degrading Pseudomonas stutzeri strains. In these strains both toluene degradation pathways (TMO and Xyl) are active and induced by toluene and phenol. The whole genome sequence of the representative strain 2A20 revealed the presence of complete TMO- and Xyl-upper pathway operons together with two sets of lower catechol meta pathway operons, as well as phenol-degrading operon in a single 292,430bp contig. The much lower GC content and analysis of the predicted ORFs refer to the plasmid origin of the approximately 130kb region of this contig, containing the xyl, phe and tou genes. The deduced amino acid sequences of the TMO, XylA and the large subunit of phenol monooxygenase (LmPH) show 98-100% identity with the respective gene products of the strain Pseudomonas sp. OX1. In both strains 2A20 and OX1 the meta-cleavage pathways for catechol degradation are coded by two redundant operons (phe and xyl). We show that in the strain 2A20 TouR and XylR are activated by different effector molecules, phenol and toluene, respectively, and they both control transcription of the xyl upper, tou (TMO) and phe catabolic operons. Although the growth parameters of redundant strains did not show advantage at toluene biodegradation, the functional redundancy could provide better flexibility to the bacteria in environmental conditions. PMID:27185632

  12. Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation.

    PubMed

    Suen, W C; Spain, J C

    1993-03-01

    The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons. PMID:8449889

  13. The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

    PubMed Central

    Pizzo, Elio; Notomista, Eugenio; Pezzella, Alessandro; Di Cristo, Carlo; De Lise, Federica; Di Donato, Alberto; Izzo, Viviana

    2015-01-01

    Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules. PMID:25915063

  14. Toluene Monooxygenase-Catalyzed Epoxidation of Alkenes

    PubMed Central

    McClay, Kevin; Fox, Brian G.; Steffan, Robert J.

    2000-01-01

    Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 0.33 μmol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified. PMID:10788354

  15. Some properties of a soluble methane mono-oxygenase from Methylococcus capsulatus strain Bath.

    PubMed Central

    Colby, J; Dalton, H

    1976-01-01

    Soluble extracts of Methylococcus capsulatus (Bath), obtained by centrifugation of crude extracts at 160000g for 1h, catalyse the NAD(P)H- and O2-dependent disappearance of bromomethane, and also the formation of methanol from methane. Soluble methane mono-oxygenase is not inhibited by chelating agents or by most electron-transport inhibitors, and is a multicomponent enzyme. PMID:962879

  16. Electron transfer control in soluble methane monooxygenase.

    PubMed

    Wang, Weixue; Iacob, Roxana E; Luoh, Rebecca P; Engen, John R; Lippard, Stephen J

    2014-07-01

    The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism. PMID:24937475

  17. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOMPONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    EPA Science Inventory

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. ix open reading frames were identified, four of which are, homologous to the components of toluene dioxy...

  18. Hydroxylation of methane through component interactions in soluble methane monooxygenases.

    PubMed

    Lee, Seung Jae

    2016-04-01

    Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system and recent applications of methane gas in the field of bioenergy and bioremediation. Methanotropic bacteria perform a specific microbial conversion from methane, one of the most stable carbon compounds, to methanol through elaborate mechanisms. MMOs express particulate methane monooxygenase (pMMO) in most strains and soluble methane monooxygenase (sMMO) under copper-limited conditions. The mechanisms of MMO have been widely studied from sMMO belonging to the bacterial multicomponent monooxygenase (BMM) superfamily. This enzyme has diiron active sites where different types of hydrocarbons are oxidized through orchestrated hydroxylase, regulatory and reductase components for precise control of hydrocarbons, oxygen, protons, and electrons. Recent advances in biophysical studies, including structural and enzymatic achievements for sMMO, have explained component interactions, substrate pathways, and intermediates of sMMO. In this account, oxidation of methane in sMMO is discussed with recent progress that is critical for understanding the microbial applications of C-H activation in one-carbon substrates. PMID:27033202

  19. Multicomponent membranes

    DOEpatents

    Kulprathipanja, Santi; Kulkarni, Sudhir S.; Funk, Edward W.

    1988-01-01

    A multicomponent membrane which may be used for separating various components which are present in a fluid feed mixture comprises a mixture of a plasticizer such as a glycol and an organic polymer cast upon a porous organic polymer support. The membrane may be prepared by casting an emulsion or a solution of the plasticizer and polymer on the porous support, evaporating the solvent and recovering the membrane after curing.

  20. Structural biology of heme monooxygenases

    SciTech Connect

    Poulos, Thomas L. . E-mail: poulos@uci.edu

    2005-12-09

    Over the past few years the number of crystal structures available for heme monooxygenases has substantially increased. Those most closely related to one another are cytochrome P450, nitric oxide synthase, and heme oxygenase. The present mini-review provides a summary of some recently published work on how crystallography and solution studies have provided new insights on function and especially the oxygen activation process. It now appears that in all three monooxygenases highly ordered solvent in the active site serves as direct proton donors to the iron-linked dioxygen; a requirement for splitting the O-O bond. This is in sharp contrast to the related peroxidase family of enzymes where strategically positioned amino acid side chains serve the function of shuttling protons. The P450cam-oxy-complex as well as various mutants in a complex with either oxygen or carbon monoxide have enabled a fairly detailed picture to be developed on the role of specific amino acids and conformational changes in both electron transfer and oxygen activation.

  1. Squalene monooxygenase - a target for hypercholesterolemic therapy.

    PubMed

    Belter, Agnieszka; Skupinska, Miroslawa; Giel-Pietraszuk, Malgorzata; Grabarkiewicz, Tomasz; Rychlewski, Leszek; Barciszewski, Jan

    2011-12-01

    Squalene monooxygenase catalyzes the epoxidation of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compounds of these organisms and squalene epoxidation is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively, which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clinical use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clinical practice. After many years of use of statins there is some clinical evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, especially in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the

  2. ASSAY FOR DETECTION AD ENUMERATION OF GENETICALLY ENGINEERED MICROORGANISMS WHICH IS BASED ON THE ACTIVITY OF A DEREGULATED 2,4-DICHLOROPHENOXYACETATE MONOOXYGENASE

    EPA Science Inventory

    An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which coverts phenoxyacetate (PAA) to phenol. n PAA-amended cultures of Pseudomonas aeruginosa PAO1C (pRO103) and Ps...

  3. Cellulose degradation by polysaccharide monooxygenases.

    PubMed

    Beeson, William T; Vu, Van V; Span, Elise A; Phillips, Christopher M; Marletta, Michael A

    2015-01-01

    Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity. PMID:25784051

  4. Electron-transfer reactions and functionalization of cytochrome P450cam monooxygenase system in reverse micelles.

    PubMed

    Ichinose, Hirofumi; Michizoe, Junji; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro

    2004-06-22

    Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media. PMID:15986701

  5. Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase

    PubMed Central

    2015-01-01

    The multicomponent protein toluene/o-xylene monooxygenase (ToMO) activates molecular oxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, two electrons are injected into each of two diiron(III) units of the hydroxylase, a process that involves three redox active proteins: the ToMO hydroxylase (ToMOH), Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). In addition to these three proteins, a small regulatory protein is essential for catalysis (ToMOD). Through steady state and pre-steady state kinetics studies, we show that ToMOD attenuates electron transfer from ToMOC to ToMOH in a concentration-dependent manner. At substoichiometric concentrations, ToMOD increases the rate of turnover, which we interpret to be a consequence of opening a pathway for oxygen transport to the catalytic diiron center in ToMOH. Excess ToMOD inhibits steady state catalysis in a manner that depends on ToMOC concentration. Through rapid kinetic assays, we demonstrate that ToMOD attenuates formation of the ToMOC–ToMOH complex. These data, coupled with protein docking studies, support a competitive model in which ToMOD and ToMOC compete for the same binding site on the hydroxylase. These results are discussed in the context of other studies of additional proteins in the superfamily of bacterial multicomponent monooxygenases. PMID:25402597

  6. NMR structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath).

    PubMed

    Chatwood, Lisa L; Müller, Jens; Gross, John D; Wagner, Gerhard; Lippard, Stephen J

    2004-09-28

    Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases. PMID:15379538

  7. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  8. Comparison of Bacillus monooxygenase genes for unique fatty acid production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reviews Bacillus genes encoding monooxygenase enzymes producing unique fatty acid metabolites. Specifically, it examines standard monooxygenase electron transfer schemes and related domain structures of these fused domain enzymes on route to understanding the observed oxygenase activiti...

  9. Starch-degrading polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Marletta, Michael A

    2016-07-01

    Polysaccharide degradation by hydrolytic enzymes glycoside hydrolases (GHs) is well known. More recently, polysaccharide monooxygenases (PMOs, also known as lytic PMOs or LPMOs) were found to oxidatively degrade various polysaccharides via a copper-dependent hydroxylation. PMOs were previously thought to be either GHs or carbohydrate binding modules (CBMs), and have been re-classified in carbohydrate active enzymes (CAZY) database as auxiliary activity (AA) families. These enzymes include cellulose-active fungal PMOs (AA9, formerly GH61), chitin- and cellulose-active bacterial PMOs (AA10, formerly CBM33), and chitin-active fungal PMOs (AA11). These PMOs significantly boost the activity of GHs under industrially relevant conditions, and thus have great potential in the biomass-based biofuel industry. PMOs that act on starch are the latest PMOs discovered (AA13), which has expanded our perspectives in PMOs studies and starch degradation. Starch-active PMOs have many common structural features and biochemical properties of the PMO superfamily, yet differ from other PMO families in several important aspects. These differences likely correlate, at least in part, to the differences in primary and higher order structures of starch and cellulose, and chitin. In this review we will discuss the discovery, structural features, biochemical and biophysical properties, and possible biological functions of starch-active PMOs, as well as their potential application in the biofuel, food, and other starch-based industries. Important questions regarding various aspects of starch-active PMOs and possible economical driving force for their future studies will also be highlighted. PMID:27170366

  10. Multicomponent mass transfer

    SciTech Connect

    Taylor, R.; Krishna, R.

    1993-01-01

    This is an important book on multicomponent mass transfer, meant for readers already acquainted with the theory of mass transfer and the fundamentals of transport phenomena. Part 1, entitled Molecular Diffusion, contains the following chapters: Preliminary Concepts; The Maxwell-Stefan Relations; Fick's Law; Estimation of Diffusion Coefficients; Solution of multicomponent Diffusion Problems: The Linearized Theory; and Solution of Multicomponent Diffusion Problems: Effective Diffusivity Methods. Part 2, entitled Interphase Transfer, contains the following chapters: Mass-Transfer Coefficients; Film Theory; Unsteady-State Mass-Transfer Models; Mass Transfer in Turbulent Flow; and Simultaneous Mass and Energy Transfer. Part 3, entitled Design, contains the following chapters: Multicomponent Distillation: Mass-Transfer Models; Multicomponent Distillation: Efficiency Models; Multicomponent Distillation: A Nonequilibrium Stage Model; and Condensation of Vapor Mixtures. Appendices are provided on matrix algebra, equation-solving and estimation of a thermodynamic derivative matrix. A computer diskette is provided with the book; the examples in Chapters 1--13 are solvable using this diskette and the commercial package Mathcad which the user must obtain. A separate software package, Chemsep, is needed for some of the exercises in Chapter 14.

  11. Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.

    PubMed

    Hamamura, N; Yeager, C M; Arp, D J

    2001-11-01

    Alkane monooxygenases in Nocardioides sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C(2) to C(16). Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from Pseudomonas oleovorans were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C(6). These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8. PMID:11679317

  12. The Origin and Evolution of Baeyer—Villiger Monooxygenases (BVMOs): An Ancestral Family of Flavin Monooxygenases

    PubMed Central

    Mascotti, Maria Laura; Lapadula, Walter Jesús; Juri Ayub, Maximiliano

    2015-01-01

    The Baeyer—Villiger Monooxygenases (BVMOs) are enzymes belonging to the “Class B” of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga) and Haptophyta (Emiliania huxleyi) for the first time. Furthermore, a search for other “Class B” monooxygenases (flavoprotein monooxygenases –FMOs – and N-hydroxylating monooxygenases – NMOs) was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all “Class B” monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA) and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes. PMID:26161776

  13. Efficient multicomponent fuel algorithm

    NASA Astrophysics Data System (ADS)

    Torres, D. J.; O'Rourke, P. J.; Amsden, A. A.

    2003-03-01

    We derive equations for multicomponent fuel evaporation in airborne fuel droplets and wall films, and implement the model into KIVA-3V. Temporal and spatial variations in liquid droplet composition and temperature are not modelled but solved for by discretizing the interior of the droplet in an implicit and computationally efficient way. We find that an interior discretization is necessary to correctly compute the evolution of the droplet composition. The details of the one-dimensional numerical algorithm are described. Numerical simulations of multicomponent evaporation are performed for single droplets and compared to experimental data.

  14. Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites.

    PubMed

    Gul, Turan; Krzek, Marzena; Permentier, Hjalmar P; Fraaije, Marco W; Bischoff, Rainer

    2016-08-01

    Mammalian flavin-containing monooxygenases, which are difficult to obtain and study, play a major role in detoxifying various xenobiotics. To provide alternative biocatalytic tools to generate flavin-containing monooxygenases (FMO)-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-related to human FMOs, was tested for their ability to oxidize a set of xenobiotic compounds. For all tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, and fenbendazole], one or more monooxygenases were identified capable of converting the target compound. Chiral liquid chromatography with tandem mass spectrometry analyses of the conversions of 3-(methylthio)aniline, albendazole, and fenbendazole revealed that the respective sulfoxides are formed in good to excellent enantiomeric excess (e.e.) by several of the tested monooxygenases. Intriguingly, depending on the chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was formed. For example, when using a monooxygenase from Rhodococcus jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a 95% e.e. whereas a fungal monooxygenase yielded the respective (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases could be identified that convert the amines into their respective N-oxides. This study shows that recombinantly expressed microbial monooxygenases represent a valuable toolbox of mammalian FMO mimics that can be exploited for the production of FMO-associated xenobiotic metabolites. PMID:26984198

  15. Multicomponent Implant Releasing Dexamethasone

    NASA Astrophysics Data System (ADS)

    Nikkola, L.; Vapalahti, K.; Ashammakhi, N.

    2008-02-01

    Several inflammatory conditions are usually treated with corticosteroids. There are various problems like side effects with traditional applications of steroids, e.g. topical, or systemic routes. Local drug delivery systems have been studied and developed to gain more efficient administration with fewer side effects. Earlier, we reported on developing Dexamethasone (DX) releasing biodegradable fibers. However, their drug release properties were not satisfactory in terms of onset of drug release. Thus, we assessed the development of multicomponent (MC) implant to enhance earlier drug release from such biodegradable fibers. Poly (lactide-co-glycolide) (PLGA) and 2 wt-% and 8 wt-% DX were compounded and extruded with twin-screw extruder to form of fibers. Some of the fibers were sterilized to obtain a change in drug release properties. Four different fiber classes were studied: 2 wt-%, 8 wt-%, sterilized 2 wt-%, and sterilized 8 wt-%. 3×4 different DX-releasing fibers were then heat-pressed to form one multicomponent rod. Half of the rods where sterilized. Drug release was measured from initial fibers and multicomponent rods using a UV/VIS spectrometer. Shear strength and changes in viscosity were also measured. Drug release studies showed that drug release commenced earlier from multicomponent rods than from component fibers. Drug release from multicomponent rods lasted from day 30 to day 70. The release period of sterilized rods extended from day 23 to day 57. When compared to the original component fibers, the drug release from MC rods commenced earlier. The initial shear strength of MC rods was 135 MPa and decreased to 105 MPa during four weeks of immersion in phosphate buffer solution. Accordingly, heat pressing has a positive effect on drug release. After four weeks in hydrolysis, no disintegration was observed.

  16. [Advances in biomolecular machine: methane monooxygenases].

    PubMed

    Lu, Jixue; Wang, Shizhen; Fang, Baishan

    2015-07-01

    Methane monooxygenases (MMO), regarded as "an amazing biomolecular machine", catalyze the oxidation of methane to methanol under aerobic conditions. MMO catalyze the oxidation of methane elaborately, which is a novel way to catalyze methane to methanol. Furthermore, MMO can inspire the biomolecular machine design. In this review, we introduced MMO including structure, gene and catalytic mechanism. The history and the taxonomy of MMO were also introduced. PMID:26647577

  17. Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes.

    PubMed Central

    Xiao, Y; Heu, S; Yi, J; Lu, Y; Hutcheson, S W

    1994-01-01

    The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors. In P. syringae pv. syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion. The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold. The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P. syringae pv. phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems. HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity. A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P. aeruginosa, as well as with the related alternate sigma factors was identified within region II. Key domains are partially conserved. Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold. Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually. The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes. The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes. Images PMID:8106313

  18. Monooxygenase activity of type 3 copper proteins.

    PubMed

    Itoh, Shinobu; Fukuzumi, Shunichi

    2007-07-01

    The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism. PMID:17461541

  19. The reduced flavin-dependent monooxygenase SfnG converts dimethylsulfone to methanesulfinate.

    PubMed

    Wicht, Denyce K

    2016-08-15

    The biochemical pathway through which sulfur may be assimilated from dimethylsulfide (DMS) is proposed to proceed via oxidation of DMS to dimethylsulfoxide (DMSO) and subsequent conversion of DMSO to dimethylsulfone (DMSO2). Analogous chemical oxidation processes involving biogenic DMS in the atmosphere result in the deposition of DMSO2 into the terrestrial environment. Elucidating the enzymatic pathways that involve DMSO2 contribute to our understanding of the global sulfur cycle. Dimethylsulfone monooxygenase SfnG and flavin mononucleotide (FMN) reductase MsuE from the genome of the aerobic soil bacterium Pseudomonas fluorescens Pf0-1 were produced in Escherichia coli, purified, and biochemically characterized. The enzyme MsuE functions as a reduced nicotinamide adenine dinucleotide (NADH)-dependent FMN reductase with apparent steady state kinetic parameters of Km = 69 μM and kcat/Km = 9 min(-1) μM (-1) using NADH as the variable substrate, and Km = 8 μM and kcat/Km = 105 min(-1) μM (-1) using FMN as the variable substrate. The enzyme SfnG functions as a flavoprotein monooxygenase and converts DMSO2 to methanesulfinate in the presence of FMN, NADH, and MsuE, as evidenced by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. The results suggest that methanesulfinate is a biochemical intermediate in sulfur assimilation. PMID:27392454

  20. CRYSTALLIZATION IN MULTICOMPONENT GLASSES

    SciTech Connect

    KRUGER AA; HRMA PR

    2009-10-08

    In glass processing situations involving glass crystallization, various crystalline forms nucleate, grow, and dissolve, typically in a nonuniform temperature field of molten glass subjected to convection. Nuclear waste glasses are remarkable examples of multicomponent vitrified mixtures involving partial crystallization. In the glass melter, crystals form and dissolve during batch-to-glass conversion, melter processing, and product cooling. Crystals often agglomerate and sink, and they may settle at the melter bottom. Within the body of cooling glass, multiple phases crystallize in a non-uniform time-dependent temperature field. Self-organizing periodic distribution (the Liesegnang effect) is common. Various crystallization phenomena that occur in glass making are reviewed.

  1. Electrohydrodynamics Of Multicomponent Vesicles

    NASA Astrophysics Data System (ADS)

    Gera, Prerna; Salac, David

    2015-11-01

    The addition of cholesterol into a lipid membrane induces the formation of distinct domains. These domains try to minimize the overall energy of the system by coalescence and migration. The application of electric fields will induce flow of these membrane domains and influence the rate at which they coarsen. In this work the electrohydrodynamics of multicomponent vesicles is numerically modelled. The method uses a Cahn-Hilliard-Cook model of the lipid domains restricted to a deforming three-dimensional vesicle and will be briefly discussed. Sample results will be presented and compared to experimental observations. This work supported by NSF Grant #1253739.

  2. Magnetization of multicomponent ferrofluids.

    PubMed

    Szalai, I; Dietrich, S

    2011-08-17

    The solution of the mean spherical approximation (MSA) integral equation for isotropic multicomponent dipolar hard sphere fluids without external fields is used to construct a density functional theory (DFT), which includes external fields, in order to obtain an analytical expression for the external field dependence of the magnetization of ferrofluidic mixtures. This DFT is based on a second-order Taylor series expansion of the free energy density functional of the anisotropic system around the corresponding isotropic MSA reference system. The ensuing results for the magnetic properties are in quantitative agreement with our canonical ensemble Monte Carlo simulation data presented here. PMID:21795777

  3. Structural basis of kynurenine 3-monooxygenase inhibition.

    PubMed

    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. PMID:23575632

  4. Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides.

    PubMed

    Toda, Hiroshi; Imae, Ryouta; Komio, Tomoko; Itoh, Nobuya

    2012-10-01

    Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced (S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding (S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration. PMID:22258641

  5. Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase.

    PubMed

    Bailey, Lucas J; Elsen, Nathaniel L; Pierce, Brad S; Fox, Brian G

    2008-01-01

    Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations. PMID:17964805

  6. Trichloroethylene degradation using recombinant bacteria expressing the soluble methane monooxygenase from methylosinus trichosporium OB3b

    SciTech Connect

    Jahng, D.; Kim, C.; Wood, T.K.

    1995-12-01

    Soluble methane monooxygenase (sMMO) from M. trichosporium OB3b has the ability to degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. For efficient trichloroethylene (TCE) degradation in a foreign host, efforts are being made to improve inconsistent and low sMMO activity of the recombinant strain constructed previously (Pseudomonas putida F1/pSMMO20). Additional smmo-containing recombinant strains have been constructed including various Pseudomonas, Agrobacterium, and Rhizobium strains. Recombinant facultative methylotrophs containing the smmo locus were also constructed through electroporation and tri-parental mating using a new plasmid pSMMO50. TCE degradation by these recombinant strains was examined. The effect of metal ions on in vitro sMMO activity was also discerned to optimize the expression medium. Among the metal ions examined, Cu(I), Cu(II), Ni(II), and Zn(II) inhibited sMMO purified from trichosporium OB3b, and the effect of the metal ions on each of the components of sMMO will also be discussed. In addition, the post-segregational killing locus (hok/sok) from E. coli plasmid R1 was inserted downstream of the smmo locus to stabilize the recombinant plasmid in these host cells, and chemostat cultures were used to optimize expression of active sMMO by varying the growth rate.

  7. Substrate radical intermediates in soluble methane monooxygenase

    SciTech Connect

    Liu Aimin; Jin Yi; Zhang Jingyan; Brazeau, Brian J.; Lipscomb, John D. . E-mail: lipsc001@umn.edu

    2005-12-09

    EPR spin-trapping experiments were carried out using the three-component soluble methane monooxygenase (MMO). Spin-traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), {alpha}-4-pyridyl-1-oxide N-tert-butylnitrone (POBN), and nitrosobenzene (NOB) were used to investigate the possible formation of substrate radical intermediates during catalysis. In contrast to a previous report, the NADH-coupled oxidations of various substrates did not produce any trapped radical species when DMPO or POBN was present. However, radicals were detected by these traps when only the MMO reductase component and NADH were present. DMPO and POBN were found to be weak inhibitors of the MMO reaction. In contrast, NOB is a strong inhibitor for the MMO-catalyzed nitrobenzene oxidation reaction. When NOB was used as a spin-trap in the complete MMO system with or without substrate, EPR signals from an NOB radical were detected. We propose that a molecule of NOB acts simultaneously as a substrate and a spin-trap for MMO, yielding the long-lived radical and supporting a stepwise mechanism for MMO.

  8. EVALUATING MULTICOMPONENT COMPETITIVE ADSORPTION IN FIXED BEDS

    EPA Science Inventory

    An equilibrium column model (ECM) was developed to evaluate multicomponent competition in fixed-bed adsorption columns. The model ignores mass transfer resistances and uses ideal adsorbed solution theory to predict the competitive effects in multicomponent mixtures. The bed capac...

  9. Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

    SciTech Connect

    Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle; Gassner, George T.; Rosenzweig, Amy C.

    2010-07-23

    Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.

  10. Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP▿ †

    PubMed Central

    Tischler, Dirk; Eulberg, Dirk; Lakner, Silvia; Kaschabek, Stefan R.; van Berkel, Willem J. H.; Schlömann, Michael

    2009-01-01

    Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120. PMID:19482928

  11. Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3.

    PubMed Central

    Weber, F J; van Berkel, W J; Hartmans, S; de Bont, J A

    1992-01-01

    Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone. Images PMID:1315734

  12. Electron transfer reactions in the alkene mono-oxygenase complex from Nocardia corallina B-276.

    PubMed Central

    Gallagher, S C; Cammack, R; Dalton, H

    1999-01-01

    Nocardia corallina B-276 possesses a multi-component enzyme, alkene mono-oxygenase (AMO), that catalyses the stereoselective epoxygenation of alkenes. The reductase component of this system has been shown by EPR and fluorescence spectroscopy to contain two prosthetic groups, an FAD centre and a [2Fe-2S] cluster. The role of these centres in the epoxygenation reaction was determined by midpoint potential measurements and electron transfer kinetics. The order of potentials of the prosthetic groups of the reductase were FAD/FAD.=-216 mV, [2Fe-2S]/[2Fe-2S].=-160 mV and FAD./FAD.=-134 mV. Combined, these data implied that the reductase component supplied the energy required for the epoxygenation reaction and allowed a prediction of the mechanism of electron transfer within the AMO complex. The FAD moiety was reduced by bound NADH in a two-electron reaction. The electrons were then transported to the [2Fe-2S] centre one at a time, which in turn reduced the di-iron centre of the epoxygenase. Reduction of the di-iron centre is required for oxygen binding and substrate oxidation. PMID:10085230

  13. Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

    PubMed

    Chen, Yao-Sheng; Luo, Wen-I; Yang, Chung-Ling; Tu, Yi-Jung; Chang, Chun-Wei; Chiang, Chih-Hsiang; Chang, Chi-Yao; Chan, Sunney I; Yu, Steve S-F

    2014-05-01

    The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions. PMID:24629413

  14. Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase

    PubMed Central

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-01-01

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. l-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of l-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from l-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L l-lysine in 12 h. Because l-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate. PMID:25012259

  15. Characterization of a tryptophan 2-monooxygenase gene from Puccinia graminis f. sp. tritici involved in auxin biosynthesis and rust pathogenicity.

    PubMed

    Yin, Chuntao; Park, Jeong-Jin; Gang, David R; Hulbert, Scot H

    2014-03-01

    The plant hormone indole-3-acetic acid (IAA) is best known as a regulator of plant growth and development but its production can also affect plant-microbe interactions. Microorganisms, including numerous plant-associated bacteria and several fungi, are also capable of producing IAA. The stem rust fungus Puccinia graminis f. sp. tritici induced wheat plants to accumulate auxin in infected leaf tissue. A gene (Pgt-IaaM) encoding a putative tryptophan 2-monooxygenase, which makes the auxin precursor indole-3-acetamide (IAM), was identified in the P. graminis f. sp. tritici genome and found to be expressed in haustoria cells in infected plant tissue. Transient silencing of the gene in infected wheat plants indicated that it was required for full pathogenicity. Expression of Pgt-IaaM in Arabidopsis caused a typical auxin expression phenotype and promoted susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. PMID:24350783

  16. Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics.

    PubMed

    Yanık-Yıldırım, K Cansu; Vardar-Schara, Gönül

    2014-11-01

    A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl(-)/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively. PMID:25016343

  17. Pseudomonas 2007 Meeting Review

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas is an important genus of bacteria. Pseudomonas aeruginosa is the third most common nosocomial pathogen in our society, associated with chronic and eventually fatal lung disease in cystic fibrosis patients, while Pseudomonas syringae species are prominent plant pathogens. The fluorescen...

  18. Regulation of cytochrome P-450 monooxygenases in the mouse

    SciTech Connect

    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 16..cap alpha..-carbonitrile (PCN) and (4) the binding of (/sup 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 2..cap alpha..-, 6..beta..- and 15..beta..- 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 (/sup 3/H) TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of (/sup 3/H) TCPOBOP to cytosolic marcomolecular elements.

  19. Multi-Component Dark Matter

    SciTech Connect

    Zurek, Kathryn M.

    2008-11-01

    We explore multi-component dark matter models where the dark sector consists of multiple stable states with different mass scales, and dark forces coupling these states further enrich the dynamics. The multi-component nature of the dark matter naturally arises in supersymmetric models, where both R parity and an additional symmetry, such as a Z{sub 2}, is preserved. We focus on a particular model where the heavier component of dark matter carries lepton number and annihilates mostly to leptons. The heavier component, which is essentially a sterile neutrino, naturally explains the PAMELA, ATIC and synchrotron signals, without an excess in antiprotons which typically mars other models of weak scale dark matter. The lighter component, which may have a mass from a GeV to a TeV, may explain the DAMA signal, and may be visible in low threshold runs of CDMS and XENON, which search for light dark matter.

  20. Reconstitution of {beta}-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

    SciTech Connect

    Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B. . E-mail: itbvkha@po.uni-stuttgart.de

    2006-01-06

    CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards {beta}-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 {sup o}C. In this system, {beta}-carotene was hydroxylated to {beta}-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 {beta}-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated {beta}-carotene at 3- and also 3'-positions, resulting in {beta}-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol {beta}-cryptoxanthin produced per nmol P450 per min.

  1. Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100.

    PubMed Central

    Xun, L

    1996-01-01

    Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself. PMID:8626333

  2. Multicomponent reactions in nucleoside chemistry

    PubMed Central

    Buchowicz, Włodzimierz

    2014-01-01

    Summary This review covers sixty original publications dealing with the application of multicomponent reactions (MCRs) in the synthesis of novel nucleoside analogs. The reported approaches were employed for modifications of the parent nucleoside core or for de novo construction of a nucleoside scaffold from non-nucleoside substrates. The cited references are grouped according to the usually recognized types of the MCRs. Biochemical properties of the novel nucleoside analogs are also presented (if provided by the authors). PMID:25161730

  3. A Sustainable Multicomponent Pyrimidine Synthesis.

    PubMed

    Deibl, Nicklas; Ament, Kevin; Kempe, Rhett

    2015-10-14

    Since alcohols are accessible from indigestible biomass (lignocellulose), the development of novel preferentially catalytic reactions in which alcohols are converted into important classes of fine chemicals is a central topic of sustainable synthesis. Multicomponent reactions are especially attractive in organic chemistry as they allow the synthesis of large libraries of diversely functionalized products in a short time when run in a combinatorial fashion. Herein, we report a novel, regioselective, iridium-catalyzed multicomponent synthesis of pyrimidines from amidines and up to three (different) alcohols. This reaction proceeds via a sequence of condensation and dehydrogenation steps which give rise to selective C-C and C-N bond formations. While the condensation steps deoxygenate the alcohol components, the dehydrogenations lead to aromatization. Two equiv of hydrogen and water are liberated in the course of the reactions. PN5P-Ir-pincer complexes, recently developed in our laboratory, catalyze this sustainable multicomponent process most efficiently. A total of 38 different pyrimidines were synthesized in isolated yields of up to 93%. Strong points of the new protocol are its regioselectivity and thus the immediate access to pyrimidines that are highly and unsymmetrically decorated with alkyl or aryl substituents. The combination of this novel protocol with established methods for converting alcohols to nitriles now allows to selectively assemble pyrimidines from four alcohol building blocks and 2 equiv of ammonia. PMID:26414993

  4. Investigations into the Biosynthesis, Regulation, and Self-Resistance of Toxoflavin in Pseudomonas protegens Pf-5.

    PubMed

    Philmus, Benjamin; Shaffer, Brenda T; Kidarsa, Teresa A; Yan, Qing; Raaijmakers, Jos M; Begley, Tadhg P; Loper, Joyce E

    2015-08-17

    Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf-5 is capable of producing trace levels of the triazine natural product toxoflavin (1) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf-5 with deletions in specific biosynthesis genes, which led us to propose a revised biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well-characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant-pathogenic bacteria, and introduction of toxM into the toxoflavin-sensitive strain Pseudomonas syringae DC3000 results in resistance to toxoflavin. PMID:26077901

  5. Novel cyclohexane monooxygenase from Acidovorax sp. CHX100.

    PubMed

    Salamanca, Diego; Karande, Rohan; Schmid, Andreas; Dobslaw, Daniel

    2015-08-01

    Acidovorax sp. CHX100 has a remarkable ability for growth on short cycloalkanes (C5-C8) as a sole source of carbon and energy under aerobic conditions via an uncharacterized mechanism. Transposon mutagenesis of Acidovorax sp. CHX100 revealed a novel cytochrome P450 monooxygenase (CYP450chx) which catalyzed the transformation of cyclohexane to cyclohexanol. Primer walking methods categorized CYP450chx as cytochrome P450 class I taking into account its operon structure: monooxygenase, FAD oxidoreductase, and ferredoxin. CYP450chx was successfully cloned and expressed in Escherichia coli JM109. The activity of CYP450chx was demonstrated by means of the indole co-oxidation. Biotransformation capability of CYP450chx was confirmed through the catalysis of cycloalkanes (C5-C8) to their respective cyclic alcohols. PMID:25935342

  6. Methane monooxygenase: functionalizing methane at iron and copper.

    PubMed

    Sazinsky, Matthew H; Lippard, Stephen J

    2015-01-01

    Methane monooxygenases (MMOs) catalyze the conversion of methane to methanol as the first committed step in the assimilation of this hydrocarbon into biomass and energy by methanotrophs, thus playing a significant role in the biogeochemistry of this potent greenhouse gas. Two distinct enzymes, a copper-dependent membrane protein, particulate methane monooxygenase (pMMO), and an iron-dependent cytosolic protein, soluble methane monooxygenase (sMMO), carry out this transformation using large protein scaffolds that help to facilitate the timely transport of hydrocarbon, O₂, proton, and electron substrates to buried dimetallic active sites. For both enzymes, reaction of the reduced metal centers with O₂leads to intermediates that activate the relatively inert C-H bonds of hydrocarbons to yield oxidized products. Among synthetic and biological catalysts, MMOs are unique because they are the only ones known to hydroxylate methane at ambient temperatures. As a need for new industrial catalysts and green chemical transformations increases, understanding how the different MMO metal centers efficiently accomplish this challenging chemistry has become the focus of intense study. This chapter examines current understanding of the sMMO and pMMO protein structures, their methods for substrate channeling, and mechanisms for the dimetallic activation of O₂and C-H bonds. PMID:25707469

  7. Monophenol monooxygenase and lincomysin biosynthesis in Streptomyces lincolnensis.

    PubMed

    Michalik, J; Emilianowicz-Czerska, W; Switalski, L; Raczyńska-Bojanowska, K

    1975-11-01

    Monophenol monooxygenase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase EC 1.14.18.1) was studied in melanin-positive and melanin-negative mutants of Streptomyces lincolnensis NCIB 9413, varying in the lincomycin synthesizing ability. The activities of laccase and tyrosine phenol lyase (EC 4.1.99.2) are absent in this organism. The monophenol monooxygenase catalyzes hydroxylation of monophenols (K(m) and V(max) for l-tyrosine, 2 x 10(-4) M and 8.0 nmol of O(2)/min per ml, respectively) at a slower rate than it dehydrogenates diphenols to o-quinones (K(m) and V(max) for l-3,4-dihydroxyphenylalanine, 7 x 10(-5) M and 51.7 nmol of O(2)/min per ml, respectively. It is inhibited by KCN, beta-mercaptoethanol, ethylenediaminetetraacetate, dipyridyl, thiourea, p-aminobenzoic acids and by some tryptophan metabolites. Changes in the activity of monophenol monooxygenase caused by mutation or by inhibitors are reflected in the synthesis of the antibiotic. Its participation in the biogenesis of the propylhygric moiety of lincomycin is discussed. PMID:813570

  8. A Multicomponent Latent Trait Model for Diagnosis

    ERIC Educational Resources Information Center

    Embretson, Susan E.; Yang, Xiangdong

    2013-01-01

    This paper presents a noncompensatory latent trait model, the multicomponent latent trait model for diagnosis (MLTM-D), for cognitive diagnosis. In MLTM-D, a hierarchical relationship between components and attributes is specified to be applicable to permit diagnosis at two levels. MLTM-D is a generalization of the multicomponent latent trait…

  9. Pseudomonas screening assay

    NASA Technical Reports Server (NTRS)

    Margalit, Ruth (Inventor)

    1993-01-01

    A method for the detection of Pseudomonas bacteria is described where an Azurin-specific antibody is employed for detecting the presence of Azurin in a test sample. The detection of the presence of Azurin in the sample is a conclusive indicator of the presence of the Pseudomonas bacteria since the Azurin protein is a specific marker for this bacterial strain.

  10. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  11. Pseudomonas kuykendallii sp. nov.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This is a submission to the list of microorganisms with standing in nomenclature maintained by the International Journal of Systematic and Evolutionary Microbiology. We wish to have Pseudomonas kuykendallii sp. nov. added to the list as a valid species belonging to the genus Pseudomonas. Three str...

  12. Active site models for the Cu(A) site of peptidylglycine α-hydroxylating monooxygenase and dopamine β-monooxygenase.

    PubMed

    Kunishita, Atsushi; Ertem, Mehmed Z; Okubo, Yuri; Tano, Tetsuro; Sugimoto, Hideki; Ohkubo, Kei; Fujieda, Nobutaka; Fukuzumi, Shunichi; Cramer, Christopher J; Itoh, Shinobu

    2012-09-01

    A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated

  13. Enthalpy Diffusion in Multicomponent Flows

    SciTech Connect

    Cook, A W

    2008-11-12

    The enthalpy diffusion flux in the multicomponent energy equation is a well known yet frequently neglected term. It accounts for energy changes, associated with compositional changes, resulting from species diffusion. Enthalpy diffusion is important in flows where significant mixing occurs between species of dissimilar molecular weight. The term plays a critical role in preventing local violations of the entropy condition. In simulations of nonpremixed combustion, omission of the enthalpy flux can lead to anomalous temperature gradients, which may cause mixing regions to exceed ignition conditions. The term can also play a role in generating acoustic noise in turbulent mixing layers. Euler solvers that rely on numerical diffusion to mix fluids cannot accurately predict the temperature in mixed regions. On the other hand, Navier-Stokes solvers that incorporate enthalpy diffusion can provide much more accurate results.

  14. Multi-component assembly casting

    SciTech Connect

    James, Allister W.

    2015-10-13

    Multi-component vane segment and method for forming the same. Assembly includes: positioning a pre-formed airfoil component (12) and a preformed shroud heat resistant material (18) in a mold, wherein the airfoil component (12) and the shroud heat resistant material (18) each comprises an interlocking feature (24); preheating the mold; introducing molten structural material (46) into the mold; and solidifying the molten structural material such that it interlocks the pre-formed airfoil component (12) with respect to the preformed shroud heat resistant material (18) and is effective to provide structural support for the shroud heat resistant material (18). Surfaces between the airfoil component (12) and the structural material (46), between the airfoil component (12) and the shroud heat resistant material (18), and between the shroud heat resistant material (18) and the structural material (46) are free of metallurgical bonds.

  15. Phenylacetylene reversibly inhibits the phenol hydroxylase of Pseudomonas sp. CF600 at high concentrations but is oxidized at lower concentrations.

    PubMed

    Kagle, Jeanne; Hay, Anthony G

    2006-09-01

    Alkynes are mechanism-based inhibitors of several bacterial monooxygenases, including the soluble methane monooxygenase (sMMO) of Methylococcus capsulatus and the toluene o-monooxygenase (TOM) of Burkholderia cepacia G4. In this paper, we investigated the inhibition of the phenol hydroxylase of Pseudomonas sp. CF600 by the alkyne phenylacetylene. Growth of CF600 on phenol and phenol hydroxylase activity were inhibited by phenylacetylene concentrations greater than 1.0 mM. Unlike other alkynes, which irreversibly inhibit a number of monooxygenases, inhibition of phenol hydroxylase by phenylacetylene was reversible, as demonstrated by the ability of washed cells to regain phenol hydroxylase activity. Additionally, phenylacetylene was metabolized by phenol-grown cells, yielding a yellow meta-ring fission product which absorbed light maximally at 412 nm. Phenol-grown CF600 transformed phenylacetylene to hydroxyphenylacetylene and 2-hydroxy-6-oxo-octa-2,4-dien-7-ynoic acid as detected by gas chromatography--mass spectroscopy and high-performance liquid chromatography (HPLC), respectively, while neither a derivative of CF600 with a non-functional phenol hydroxylase nor wild-type CF600 grown on acetate transformed phenylacetylene. These results demonstrate that the phenol hydroxylase of CF600 has broader substrate specificity than previously reported. They also suggest that phenylacetylene acts as a competitive inhibitor rather than as a mechanism-based inhibitor of this phenol hydroxylase. PMID:16485115

  16. Uphill diffusion in multicomponent mixtures.

    PubMed

    Krishna, Rajamani

    2015-05-21

    Molecular diffusion is an omnipresent phenomena that is important in a wide variety of contexts in chemical, physical, and biological processes. In the majority of cases, the diffusion process can be adequately described by Fick's law that postulates a linear relationship between the flux of any species and its own concentration gradient. Most commonly, a component diffuses down the concentration gradient. The major objective of this review is to highlight a very wide variety of situations that cause the uphill transport of one constituent in the mixture. Uphill diffusion may occur in multicomponent mixtures in which the diffusion flux of any species is strongly coupled to that of its partner species. Such coupling effects often arise from strong thermodynamic non-idealities. For a quantitative description we need to use chemical potential gradients as driving forces. The transport of ionic species in aqueous solutions is coupled with its partner ions because of the electro-neutrality constraints; such constraints may accelerate or decelerate a specific ion. When uphill diffusion occurs, we observe transient overshoots during equilibration; the equilibration process follows serpentine trajectories in composition space. For mixtures of liquids, alloys, ceramics and glasses the serpentine trajectories could cause entry into meta-stable composition zones; such entry could result in phenomena such as spinodal decomposition, spontaneous emulsification, and the Ouzo effect. For distillation of multicomponent mixtures that form azeotropes, uphill diffusion may allow crossing of distillation boundaries that are normally forbidden. For mixture separations with microporous adsorbents, uphill diffusion can cause supra-equilibrium loadings to be achieved during transient uptake within crystals; this allows the possibility of over-riding adsorption equilibrium for achieving difficult separations. PMID:25761383

  17. Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134.

    PubMed Central

    Streber, W R; Timmis, K N; Zenk, M H

    1987-01-01

    Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R. H. Don, A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5. A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity. The cloned tfdA gene was also transferred to A. eutrophus JMP222, which is a cured derivative of JMP134. The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy. Pseudomonas sp. strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid. Gene tfdA was subcloned and analyzed by deletions. Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments. A 2-kilobase subfragment containing tfdA has been sequenced. Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis. Images PMID:3036764

  18. Mechanistic Studies on the Hydroxylation of Methane by Methane Monooxygenase

    SciTech Connect

    Baik, Mu-Hyun; Newcomb, Martin; Friesner, Richard A.; Lippard, Stephen J.

    2003-05-17

    Methanotrophs are bacteria that live on methane as their only source of carbon.1 The first step in their utilization of this simplest of all hydrocarbons is its selective conversion to methanol. Subsequent biochemical pathways transform methanol to formaldehyde, which in turn is processed into biomass. Further oxidation of formaldehyde to carbon dioxide provides energy that is stored for later use as NADH.2 The conversion of methane to methanol is catalyzed at the active site of a metalloenzyme known as methane monooxygenase, or MMO.3-9

  19. On the catalytic mechanisms of lytic polysaccharide monooxygenases.

    PubMed

    Walton, Paul H; Davies, Gideon J

    2016-04-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper-containing oxygenases. LPMOs oxidise recalcitrant polysaccharides such as chitin and cellulose, thereby making these substrates more tractable to canonical chitinase or cellulase action. As such, LPMOs are attracting much attention not only for their capacity to greatly increase the efficiency of production of cellulosic-based biofuels, but also for the new questions they pose about the mechanisms of biological oxidation of recalcitrant substrates. This review draws together the current thinking on the catalytic mechanisms of LPMOs and other copper catalysed oxygenations and provides a blueprint for further investigation into the mechanisms of action of these intriguing enzymes. PMID:27094791

  20. The framework of polysaccharide monooxygenase structure and chemistry.

    PubMed

    Span, Elise A; Marletta, Michael A

    2015-12-01

    Polysaccharide monooxygenases, or PMOs (also known as lytic PMOs or LPMOs), are a group of enzymes discovered in recent years to catalyze the oxidative degradation of carbohydrate polymers. The PMO catalytic domain has a β-sandwich fold that bears a strong resemblance to both immunoglobulin (Ig) and fibronectin type III (FnIII) domains. PMOs are secreted by fungi and bacteria, and there is recent evidence for their roles in pathogenesis, in addition to biomass processing. This review addresses the biological origins and functions of emerging PMO families, as well as describes the aspects of PMO structure that support the chemistry of copper-catalyzed, oxidative polysaccharide degradation. PMID:26615470

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

    PubMed

    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. PMID:24316190

  2. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

    PubMed

    Frandsen, Kristian E H; Simmons, Thomas J; Dupree, Paul; Poulsen, Jens-Christian N; Hemsworth, Glyn R; Ciano, Luisa; Johnston, Esther M; Tovborg, Morten; Johansen, Katja S; von Freiesleben, Pernille; Marmuse, Laurence; Fort, Sébastien; Cottaz, Sylvain; Driguez, Hugues; Henrissat, Bernard; Lenfant, Nicolas; Tuna, Floriana; Baldansuren, Amgalanbaatar; Davies, Gideon J; Lo Leggio, Leila; Walton, Paul H

    2016-04-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains. PMID:26928935

  3. The metal centres of particulate methane mono-oxygenase.

    PubMed

    Rosenzweig, Amy C

    2008-12-01

    pMMO (particulate methane mono-oxygenase) is an integral membrane metalloenzyme that catalyses the oxidation of methane to methanol. The pMMO metal active site has not been identified, precluding detailed investigation of the reaction mechanism. Models for the metal centres proposed by various research groups have evolved as crystallographic and spectroscopic data have become available. The present review traces the evolution of these active-site models before and after the 2005 Methylococcus capsulatus (Bath) pMMO crystal structure determination. PMID:19021511

  4. Laser ultrasonic multi-component imaging

    DOEpatents

    Williams, Thomas K.; Telschow, Kenneth

    2011-01-25

    Techniques for ultrasonic determination of the interfacial relationship of multi-component systems are discussed. In implementations, a laser energy source may be used to excite a multi-component system including a first component and a second component at least in partial contact with the first component. Vibrations resulting from the excitation may be detected for correlation with a resonance pattern indicating if discontinuity exists at the interface of the first and second components.

  5. Multicomponent fuel vaporization at high pressures.

    SciTech Connect

    Torres, D. J.; O'Rourke, P. J.

    2002-01-01

    We extend our multicomponent fuel model to high pressures using a Peng-Robinson equation of state, and implement the model into KIVA-3V. Phase equilibrium is achieved by equating liquid and vapor fugacities. The latent heat of vaporization and fuel enthalpies are also corrected for at high pressures. Numerical simulations of multicomponent evaporation are performed for single droplets for a diesel fuel surrogate at different pressures.

  6. Indicator For Pseudomonas Bacteria

    NASA Technical Reports Server (NTRS)

    Margalit, Ruth

    1990-01-01

    Characteristic protein extracted and detected. Natural protein marker found in Pseudomonas bacteria. Azurin, protein containing copper readily extracted, purified, and used to prepare antibodies. Possible to develop simple, fast, and accurate test for marker carried out in doctor's office.

  7. Role of flavin-containing monooxygenase in drug development.

    PubMed

    Cashman, John R

    2008-12-01

    This review summarizes some recent observations and information related to the role of the flavin-containing monooxygenase (FMO) in preclinical drug development. Flavin-containing monooxygenase is a complimentary enzyme system to the cytochrome P450 (CYP) family of enzymes and oxygenates several soft, highly polarizable nucleophilic heteroatom-containing chemicals and drugs. The products of FMO-mediated metabolism are generally benign and highly polar, readily excreted materials. There may be some advantages in designing drugs that are metabolized in part by FMO and not exclusively by CYP. In this review, I describe the practical aspects for the participation of FMO in drug and chemical metabolism including: i) the study of FMO using in vitro preparations; ii) some observations about metabolism of drugs and chemicals by FMO in vivo; and iii) the consequences of studying FMO-related metabolism in various small animal models. Some of the preclinical research and development areas related to FMO are not fully mature areas and there are certain gaps in our knowledge. However, I include discussion of these areas to stimulate further work and invite further discussion. PMID:19040327

  8. The methane monooxygenase intrinsic activity of kinds of methanotrophs.

    PubMed

    Zhang, Yingxin; Xin, Jiaying; Chen, Linlin; Xia, Chungu

    2009-06-01

    Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1-5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs. PMID:19052919

  9. 40 CFR 180.1114 - Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS; exemptions from the requirement of a tolerance... Tolerances § 180.1114 Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas...

  10. 40 CFR 180.1114 - Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS; exemptions from the requirement of a tolerance... Tolerances § 180.1114 Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas...

  11. 40 CFR 180.1114 - Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS; exemptions from the requirement of a tolerance... Tolerances § 180.1114 Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas...

  12. 40 CFR 180.1114 - Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS; exemptions from the requirement of a tolerance... Tolerances § 180.1114 Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas...

  13. 40 CFR 180.1114 - Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas syringae 742RS; exemptions from the requirement of a tolerance... Tolerances § 180.1114 Pseudomonas fluorescens A506, Pseudomonas fluorescens 1629RS, and Pseudomonas...

  14. Diclofenac Sodium Loaded Multicomponent Implant

    NASA Astrophysics Data System (ADS)

    Nikkola, Lila; Viitanen, Petrus; Ashammakhi, Nureddin

    2008-02-01

    Earlier we have reported on developing DS releasing bioabsorbable rods for inhibition of osteolysis [l]. Due to their unsatisfactory drug release profiles we assessed the use of sintering technique of enhancement of drug release in the current study. Melt extruded PLGA 80/20 rods were compounded 8 wt-% DS. Some rods were self reinforced (SR) and some of them were sterilized to get three different components with different drug release profiles. Different rods were sintered together with heat and pressure. Three different specimen groups with different construction were studied. Thermal properties were analyzed using differential scanning calorimetry (DSC). Changes of IV were performed with capillary analysis and drug release measurements with UV-Vis spectrophotometer. Mechanical strength were measured two weeks, when disintegration occurred. Release rate consisted of 1) sharp jump start peak, 2) second smoother peak, and 3) third smooth peak. Released DS concentrations reached local therapeutic levels and maintained at that stage for 24-36 days. All DS was released during 50-70 days. The drug release from multicomponent implant was more stable and commenced earlier than from initial rods. Such properties were favored ones. Initial shear strength was 82 MPa and it decreased to 15 MPa. The mechanical bonding was sufficient although the components disintegrated relatively fast. By sintering different PLGA/DS components with different release rates it is possible to construct a truly controlled release implant for bone fixation with anti-inflammatory properties.

  15. Dynamics in Multicomponent Polyelectrolyte Solutions

    NASA Astrophysics Data System (ADS)

    Nagao, Michihiro

    2010-03-01

    Double-network hydrogels (DN-gel) prepared from the combination of a moderately cross-linked anionic polyelectrolyte (poly(2-acrylamido-2-methyl-1-propanesulfonic acid), PAMPS) and an un-cross-linked linear polymer (polyacrylamide, PAAm) solution show strong mechanical properties far superior to that of their individual constituents [1]. To determine the origin of the superior properties of DN-gels, we investigated the structure and the chain dynamics of model PAMPS/PAAm solution blends using small-angle neutron scattering and neutron spin-echo measurements [2]. Akcasu's dynamic scattering theory for a multicomponent system [3] is modified to include polyelectrolytes, and the resulting equation describes well the neutron spin-echo results over the entire wavevector range covered in our experiments. Parameters such as effective solvent viscosity were deduced from the measured data using the modified Akcasu equation. Both the relaxation time at large length scales (10-100 nm) and the segmental diffusion coefficient at short length scales (0.1-1 nm) or the effective solvent viscosity show good accordance with the macroscopic rheological behavior of the solution blends. [4pt] [1] J.P. Gong et al., Adv. Mater. 15, 1155 (2003). [0pt] [2] S. Lee et al., Macromolecules 42, 1293 (2009). [0pt] [2] A.Z. Akcasu, in Dynamic Liht Scattering, The Method and Some Applications; W. Brown Ed. (Oxford University Press, London 1992).

  16. Crystallization of classical multicomponent plasmas

    SciTech Connect

    Medin, Zach; Cumming, Andrew

    2010-03-15

    We develop a method for calculating the equilibrium properties of the liquid-solid phase transition in a classical, ideal, multicomponent plasma. Our method is a semianalytic calculation that relies on extending the accurate fitting formulas available for the one-, two-, and three-component plasmas to the case of a plasma with an arbitrary number of components. We compare our results to those of C. J. Horowitz et al. [Phys. Rev. E 75, 066101 (2007)], who used a molecular-dynamics simulation to study the chemical properties of a 17-species mixture relevant to the ocean-crust boundary of an accreting neutron star at the point where half the mixture has solidified. Given the same initial composition as Horowitz et al., we are able to reproduce to good accuracy both the liquid and solid compositions at the half-freezing point; we find abundances for most species within 10% of the simulation values. Our method allows the phase diagram of complex mixtures to be explored more thoroughly than possible with numerical simulations. We briefly discuss the implications for the nature of the liquid-solid boundary in accreting neutron stars.

  17. Multicomponent Therapeutics of Berberine Alkaloids

    PubMed Central

    Luo, Jiaoyang; Yan, Dan; Yang, Meihua; Dong, Xiaoping; Xiao, Xiaohe

    2013-01-01

    Although berberine alkaloids (BAs) are reported to be with broad-spectrum antibacterial and antiviral activities, the interactions among BAs have not been elucidated. In the present study, methicillin-resistant Staphylococcus aureus (MRSA) was chosen as a model organism, and modified broth microdilution was applied for the determination of the fluorescence absorption values to calculate the anti-MRSA activity of BAs. We have initiated four steps to seek the optimal combination of BAs that are (1) determining the anti-MRSA activity of single BA, (2) investigating the two-component combination to clarify the interactions among BAs by checkerboard assay, (3) investigating the multicomponent combination to determine the optimal ratio by quadratic rotation-orthogonal combination design, and (4) in vivo and in vitro validation of the optimal combination. The results showed that the interactions among BAs are related to their concentrations. The synergetic combinations included “berberine and epiberberine,” “jatrorrhizine and palmatine” and “jatrorrhizine and coptisine”; the antagonistic combinations included “coptisine and epiberberine”. The optimal combination was berberine : coptisine : jatrorrhizine : palmatine : epiberberine = 0.702 : 0.863 : 1 : 0.491 : 0.526, and the potency of the optimal combination on cyclophosphamide-immunocompromised mouse model was better than the natural combinations of herbs containing BAs. PMID:23634170

  18. Hot Tub Rash (Pseudomonas Folliculitis)

    MedlinePlus

    ... rash and rashes clinical tools newsletter | contact Share | Hot Tub Rash ( Pseudomonas Folliculitis) Information for adults A ... the skin and small pus-filled lesions. Overview Hot tub rash ( Pseudomonas folliculitis) is an infection of ...

  19. Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b.

    PubMed Central

    Tonge, G M; Harrison, D E; Higgins, I J

    1977-01-01

    1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed. Images Fig. 3. PMID:15544

  20. In silico analysis for prediction of degradative capacity of Pseudomonas putida SF1.

    PubMed

    Tikariha, Hitesh; Pal, Rajesh Ramavadh; Qureshi, Asifa; Kapley, Atya; Purohit, Hemant J

    2016-10-15

    The study employs draft genome sequence data to explore p-nitrophenol (PNP) degradation activity of Pseudomonas putida strain SF-1 at a genomic scale. Annotation analysis proposes that the strain SF1 not only possesses the gene cluster for PNP utilization but also for the utilization of benzoate, catechol, hydroxybenzoate, protocatechuate, and homogentisate. Further, the analysis was carried out to understand more details of PNP 4-monooxygenase and its regulator. A comparative analysis of PNP 4-monooxygenase from SF1 was carried out for prediction of its tertiary structure; and also its binding affinity with PNP, FAD, NADH and NADPH using FlexX docking. The tertiary structure of regulator was also predicted along with its conserved DNA binding residues. Regulator binding site (RBS) and promoter region were mapped for the PNP degradation gene cluster. Based on genome sequence analysis, the study unveiled the genomic attributes for a versatile catabolic potential of Pseudomonas putida strain SF-1 for different aromatic compounds. PMID:27317892

  1. Conscientiousness increases efficiency of multicomponent behavior

    PubMed Central

    Stock, Ann-Kathrin; Beste, Christian

    2015-01-01

    Many everyday situations require the flexible interruption and changing of different actions to achieve a goal. Several strategies can be applied to do so, but those requiring high levels of cognitive control seem to confer an efficiency (speed) advantage in situations requiring multi-component behavior. However, it is elusive in how far personality traits affect performance in such situations. Given that top-down control is an important aspect of personality and furthermore correlates with conscientiousness, N = 163 participants completed the NEO-FFI and performed an experimental (stop-change) paradigm assessing multicomponent behavior. Applying mathematical constraints to the behavioral data, we estimated the processing strategy of each individual. The results show that multicomponent behavior is selectively affected by conscientiousness which explained approximately 19% of the measured inter-individual behavioral variance. Conscientiousness should hence be seen as a major personality dimension modulating multicomponent behavior. Highly conscientious people showed a more effective, step-by-step processing strategy of different actions necessary to achieve a goal. In situations with simultaneous requirements, this strategy equipped them with an efficiency (speed) advantage towards individuals with lower conscientiousness. In sum, the results show that strategies and the efficiency with which people cope with situations requiring multicomponent behavior are strongly influenced by their personality. PMID:26503352

  2. Multicomponent liquid ion exchange with chabazite zeolites

    SciTech Connect

    Robinson, S.M.; Arnold, W.D. Jr.; Byers, C.W.

    1993-10-01

    In spite of the increasing commercial use of zeolites for binary and multicomponent sorption, the understanding of the basic mass-transfer processes associated with multicomponent zeolite ion-exchange systems is quite limited. This study was undertaken to evaluate Na-Ca-Mg-Cs-Sr ion exchange from an aqueous solution using a chabazite zeolite. Mass-transfer coefficients and equilibrium equations were determined from experimental batch-reactor data for single and multicomponent systems. The Langmuir isotherm was used to represent the equilibrium relationship for binary systems, and a modified Dubinin-Polyani model was used for the multicomponent systems. The experimental data indicate that diffusion through the microporous zeolite crystals is the primary diffusional resistance. Macropore diffusion also significantly contributes to the mass-transfer resistance. Various mass-transfer models were compared to the experimental data to determine mass-transfer coefficients. Effective diffusivities were obtained which accurately predicted experimental data using a variety of models. Only the model which accounts for micropore and macropore diffusion occurring in series accurately predicted multicomponent data using single-component diffusivities. Liquid and surface diffusion both contribute to macropore diffusion. Surface and micropore diffusivities were determined to be concentration dependent.

  3. Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Beeson, William T; Phillips, Christopher M; Cate, Jamie H D; Marletta, Michael A

    2014-01-15

    The ubiquitous fungal polysaccharide monooxygenases (PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are structurally related but have significant variation in sequence. A heterologous expression method in Neurospora crassa was developed as a step toward connecting regioselectivity of the chemistry to PMO phylogeny. Activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal PMOs fall into three major groups with distinctive active site surface features. PMO1s and PMO2s hydroxylate glycosidic positions C1 and C4, respectively. PMO3s hydroxylate both C1 and C4. A subgroup of PMO3s (PMO3*) hydroxylate C1. Mutagenesis studies showed that an extra subdomain of about 12 amino acids contribute to C4 oxidation in the PMO3 family. PMID:24350607

  4. Quantum Chemical Studies of Methane Monooxygenase: Comparison with P450

    SciTech Connect

    Guallar, Victor; Gherman, Benjamin F.; Lippard, Stephen J.; Friesner, Richard A.

    2002-04-01

    The catalytic pathways of soluble methane monooxygenase (sMMO) and cytochrome P450CAM, iron-containing enzymes, are described and compared. Recent extensive density functional ab initio electronic structure calculations have revealed many similarities in a number of the key catalytic steps, as well as some important differences. A particularly interesting and significant contrast is the role played by the protein in each system. For sMMO, the protein stabilizes various species in the catalytic cycle through a series of carboxylate shifts. This process is adequately described by a relatively compact model of the active site (similar to100 atoms), providing a reasonable description of the energetics of hydrogen atom abstraction. For P450CAM, in contrast, the inclusion of the full protein is necessary for an accurate description of the hydrogen atom abstraction.

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

    PubMed

    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. PMID:25715464

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

    PubMed

    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. PMID:26589832

  7. Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination.

    PubMed Central

    Chen, Y C; Peoples, O P; Walsh, C T

    1988-01-01

    The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli. Images PMID:3338974

  8. Aryl hydrocarbon mono-oxygenase activity in human lymphocytes

    SciTech Connect

    Griffin, G.D.; Schuresko, D.D.

    1981-06-01

    Aryl hydrocarbon mono-oxygenase (AHM), an enzyme of key importance in metabolism of xenobiotic chemicals such as polynuclear aromatic hydrocarbons (PNA), is present in human lymphocytes. Studies investing the relation of activity of AHM in human lymphocytes to parameters such as disease state, PNA exposure, in vitro mitogen stimulation, etc. have been summarized in this report. Some studies have demonstrated increased AHM activity in lymphocytes from cigarette smokers (compared to nonsmokers), and in lung cancer patients when compared to appropriate control groups. These observations are confused by extreme variability in human lymphocyte AHM activities, such variability arising from factors such as genetic variation in AHM activity, variation in in vitro culture conditions which affect AHM activity, and the problematical relationship of common AHM assays to actual PNA metabolism taking place in lymphocytes. If some of the foregoing problems can be adequately addressed, lymphocyte AHM activity could hold the promise of being a useful biomarker system for human PNA exposure.

  9. Polymicrobial Ventriculitis Involving Pseudomonas fulva

    PubMed Central

    Rebolledo, Paulina A.; Vu, Catphuong Cathy L.; Carlson, Renee Donahue; Kraft, Colleen S.; Anderson, Evan J.

    2014-01-01

    Infections due to Pseudomonas fulva remain a rare but emerging concern. A case of ventriculitis due to Enterobacter cloacae and Pseudomonas fulva following placement of an external ventricular drain is described. Similar to other reports, the organism was initially misidentified as Pseudomonas putida. The infection was successfully treated with levofloxacin. PMID:24648556

  10. Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations.

    PubMed

    Machovina, Melodie M; Usselman, Robert J; DuBois, Jennifer L

    2016-08-19

    Members of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means. PMID:27307041

  11. Effects of bromocriptine on hepatic cytochrome P-450 monooxygenase system.

    PubMed

    Moochhala, S M; Lee, E J; Hu, G T; Koh, O S; Becket, G

    1989-02-01

    We have evaluated the in vitro effects of bromocriptine (Br), on the hepatic cytochrome P-450 monooxygenase system of rats pretreated with saline phenobarbitone (PB) and beta-naphthoflavone (BNF). Br inhibited ethoxyresorufin O-dealkylase (EROD) activity in liver microsomes of rats pretreated with saline and PB but not in BNF pretreated animals. Maximum inhibition of EROD activity by Br in the microsomes of saline and PB pretreated rats were 50%-60% of the control. In contrast, a dual effect was observed on aminopyrine N-demethylase activity (APD) by Br in microsomes of saline, PB and BNF pretreated rats. At a low concentration (25 microM), Br inhibited the activity of APD to a similar extent in all pretreatment groups; however, with higher concentrations of Br (50 microM to 300 microM), enhancement of APD activity was observed. Br (300 microM) increased the APD activity to 2-3 times the control level in microsomes of rats pretreated with saline, PB or BNF. Spectral studies revealed a Type II binding of Br to cytochrome P-450 from microsomes of saline and PB pretreated rats. A reverse type I binding was observed for BNF induced microsomes. In addition, Br also enhanced NADPH cytochrome c (P-450) reductase activity to a similar extent in all pretreatment groups. These results suggest that the inhibition of EROD activity may be due to direct binding by Br to certain isozymes of cytochrome P-450 and that the enhancing effect of Br on APD activity may be in part due to the activation of the NADPH cytochrome c reductase component of the cytochrome P-450 monooxygenase system. PMID:2499727

  12. Evolving P450pyr Monooxygenase for Regio- and Stereoselective Hydroxylations.

    PubMed

    Yang, Yi; Li, Zhi

    2015-01-01

    P450pyr monooxygenase from Sphingomonas sp. HXN-200 catalysed the regio- and stereoselective hydroxylation at a non-activated carbon atom, a useful but challenging reaction in classic chemistry, with unique substrate specificity for a number of alicyclic compounds. New P450pyr mutants were developed by directed evolution with improved catalytic performance, thus significantly extending the application of the P450pyr monooxygenase family in biohydroxylation to prepare useful and valuable chiral alcohols. Directed evolution of P450pyr created new enzymes with improved S-enantioselectivity or R-enantioselectivity for the hydroxylation of N-benzyl pyrrolidine, enhanced regioselectivity for the hydroxylation of N-benzyl pyrrolidinone, and increased enantioselectivity for the hydroxylation of N-benzyl piperidinone, respectively. Directed evolution of P450pyr generated also mutants with fully altered regioselectivity (from terminal to subterminal) and newly created excellent S-enantioselectivity for the biohydroxylation of n-octane and propylbenzene, respectively, providing new opportunities for the regio- and enantioselective alkane functionalization. New P450pyr mutants were engineered as the first catalyst for highly selective terminal hydroxylation of n-butanol to 1,4-butanediol. Several novel, accurate, sensitive, simple, and HTS assays based on colorimetric or MS detection for measuring the enantio- and/or regioselectivity of hydroxylation were developed and proven to be practical in directed evolution. The P450pyr X-ray structure was obtained and used to guide the evolution. In silico modelling and substrate docking provided some insight into the influence of several important amino acid mutations of the engineered P450pyr mutants on the altered or enhanced regio- and enantioselectivity as well as new substrate acceptance. The obtained information and knowledge is useful for further engineering of P450pyr for other hydroxylations and oxidations. PMID:26507217

  13. Pseudomonas psychrotolerans sp. nov.

    PubMed

    Hauser, Elke; Kämpfer, Peter; Busse, Hans-Jürgen

    2004-09-01

    Three yellow-pigmented, Gram-negative, rod-shaped, non-spore-forming bacterial strains, C36T, C37 and C39, were isolated in the Medical Clinic for Small Animals and Ungulates at the University for Veterinary Medicine in Vienna, Austria. On the basis of 16S rRNA gene sequence similarity, strain C36T was shown to belong to the genus Pseudomonas; Pseudomonas oleovorans DSM 1045T was the nearest relative (99.5 % sequence similarity). Other Pseudomonas species shared <97 % sequence similarity with strain C36T. The presence of Q-9 as the major ubiquinone, the predominance of putrescine and spermidine in its polyamine patterns and its fatty acid profile [i.e. the predominance of C(16 : 0), summed feature 3 (C(16 : 1)omega7c and/or 2-OH C(15 : 0) iso), C(18 : 1)omega7c and the presence of 3-OH C(10 : 0), 3-OH C(12 : 0) and 2-OH C(12 : 0)] were in agreement with identification of this strain as a member of the genus Pseudomonas. Physiological and biochemical characteristics and the results of genomic fingerprinting clearly differentiated strain C36T from its phylogenetic relative P. oleovorans DSM 1045T. Results from DNA-DNA hybridization showed that strain C36T represents a species that is distinct from P. oleovorans DSM 1045T. These data demonstrate that strain C36T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas psychrotolerans sp. nov. is proposed. The type strain is C36T (= LMG 21977T = DSM 15758T). Additionally, physiological, biochemical, chemotaxonomic and genomic fingerprints indicate that P. oleovorans ATCC 29347 may not be a member of the species P. oleovorans sensu stricto. PMID:15388721

  14. Multicomponent Transport in Polyatomic Reactive Gas Mixtures

    NASA Astrophysics Data System (ADS)

    Giovangigli, Vincent

    2011-05-01

    We investigate multicomponent reactive flow models derived from the kinetic theory of gases. We discuss in particular the conservation equations, the transport fluxes and the transport coefficients in weak and strong magnetic fields. The mathematical properties of the resulting hyperbolic-parabolic systems of partial differential equations modeling multicomponent flows are deduced from the underlying kinetic framework. The structure and solution of the transport linear systems associated with the evaluation of transport coefficients are also addressed. In particular, the convergence of iterative techniques is deduced from the properties of the linearized Boltzmann collision operator. The impact of multicomponent transport is also discussed, notably the importance of Soret effects in various flows and the impact of volume viscosity.

  15. Multicomponent Transport in Polyatomic Reactive Gas Mixtures

    SciTech Connect

    Giovangigli, Vincent

    2011-05-20

    We investigate multicomponent reactive flow models derived from the kinetic theory of gases. We discuss in particular the conservation equations, the transport fluxes and the transport coefficients in weak and strong magnetic fields. The mathematical properties of the resulting hyperbolic-parabolic systems of partial differential equations modeling multicomponent flows are deduced from the underlying kinetic framework. The structure and solution of the transport linear systems associated with the evaluation of transport coefficients are also addressed. In particular, the convergence of iterative techniques is deduced from the properties of the linearized Boltzmann collision operator. The impact of multicomponent transport is also discussed, notably the importance of Soret effects in various flows and the impact of volume viscosity.

  16. Aminoazoles as Key Reagents in Multicomponent Heterocyclizations

    NASA Astrophysics Data System (ADS)

    Chebanov, Valentin A.; Gura, Katerina A.; Desenko, Sergey M.

    Because of the significant role in biological processes in living cells and the diverse types of physiological activities, heterocyclic compounds are in focus of intense investigations by academic and applied-oriented chemists. Considerably, a scientific renaissance of heterocycles during the last decades is closely related to the development of multicomponent approaches to their synthesis. Multicomponent methodology fundamentally different from two-component or sequential processes together with other innovative synthetic methods like microwave- and ultrasonic-assisted reactions offer some new possibilities in constructing heterocyclic systems with high level of molecular diversity and complexity. An overview of known multicomponent heterocyclizations using aminoazoles as a key reagent and their rich synthetic potential for obtaining five-, six-, and seven-membered heterocycles is presented. A special attention is paid to the tuning of chemo- and regio- and positional selectivity of some reactions as well as to the application of nonclassical activation methods based on microwave and ultrasonic irradiation.

  17. Cavity residue leucine 95 and channel residues glutamine 204, aspartic acid 211, and phenylalanine 269 of toluene o-xylene monooxygenase influence catalysis.

    PubMed

    Kurt, Cansu; Sönmez, Burcu; Vardar, Nurcan; Yanık-Yıldırım, K Cansu; Vardar-Schara, Gönül

    2016-09-01

    Structural analysis of toluene-o-xylene monooxygenase (ToMO) hydroxylase revealed the presence of three hydrophobic cavities, a channel, and a pore leading from the protein surface to the active site. Here, saturation mutagenesis was used to investigate the catalytic roles of alpha-subunit (TouA) second cavity residue L95 and TouA channel residues Q204, D211, and F269. By testing the substrates toluene, phenol, nitrobenzene, and/or naphthalene, these positions were found to influence the catalytic activity of ToMO. Several regiospecific variants were identified from TouA positions Q204, F269, and L95. For example, TouA variant Q204H had the regiospecificity of nitrobenzene changed significantly from 30 to 61 % p-nitrophenol. Interestingly, a combination of mutations at Q204H and A106V altered the regiospecificity of nitrobenzene back to 27 % p-nitrophenol. TouA variants F269Y, F269P, Q204E, and L95D improved the meta-hydroxylating capability of nitrobenzene by producing 87, 85, 82, and 77 % m-nitrophenol, respectively. For naphthalene oxidation, TouA variants F269V, Q204A, Q204S/S222N, and F269T had the regiospecificity changed from 16 to 9, 10, 23, and 25 % 2-naphthol, respectively. Here, two additional TouA residues, S222 and A106, were also identified that may have important roles in catalysis. Most of the isolated variants from D211 remained active, whereas having a hydrophobic residue at this position appeared to diminish the catalytic activity toward naphthalene. The mutational effects on the ToMO regiospecificity described here suggest that it is possible to further fine tune and engineer the reactivity of multicomponent diiron monooxygenases toward different substrates at positions that are relatively distant from the active site. PMID:27311562

  18. Deposition of thin films of multicomponent materials

    NASA Technical Reports Server (NTRS)

    Thakoor, Sarita (Inventor)

    1993-01-01

    Composite films of multicomponent materials, such as oxides and nitrides, e.g., lead zirconate titanate, are deposited by dc magnetron sputtering, employing a rotating substrate holder, which rotates relative to a plurality of targets, one target for each metal element of the multicomponent material. The sputtering is carried out in a reactive atmosphere. The substrates on which the layers are deposited are at ambient temperature. Following deposition of the composite film, the film is heated to a temperature sufficient to initiate a solid state reaction and form the final product, which is substantially single phase and substantially homogeneous.

  19. Multi-Component Reactions in Heterocyclic Chemistry

    NASA Astrophysics Data System (ADS)

    Müller, Thomas J. J.; Orru, Romano V. A.; Chebanov, Valentin A.; Sakhno, Yana I.; Saraev, Vyacheslav E.; Muravyova, Elena A.; Andrushchenko, Anastasia Yu.; Desenko, Sergey M.; Akhmetova, V. R.; Khabibullina, G. R.; Rakhimova, E. B.; Vagapov, R. A.; Khairullina, R. R.; Niatshina, Z. T.; Murzakova, N. N.; Maslivets, Andrey N.; Voskressensky, Leonid G.; Danagulyan, Gevorg G.; Murtchyan, Armen D.; Tumanyan, Araksya K.; Banfi, Luca; Basso, Andrea; de Moliner, Fabio; Guanti, Giuseppe; Petricci, Elena; Riva, Renata; Taddei, Maurizio; Naimi-Jamal, M. Reza; Mashkouri, Sara; Sharifi, Ali; Przhevalski, Nikolai M.; Rozhkova, Elena N.; Tokmakov, Gennadii P.; Magedov, Igor V.; Armisheva, M. N.; Rassudihina, N. A.; Vahrin, M. I.; Gein, V. L.; Shaabani, Ahmad; Rezayan, Ali Hossein; Sarvary, Afshin; Heidary, Marjan; Ng, Seik Weng; Beliaev, Nikolai A.; Mokrushin, Vladimir S.; Paramonov, Igor V.; Ilyin, Alexey; Garkushenko, Anna K.; Dushek, Maria A.; Sagitullina, Galina P.; Sagitullin, Reva S.; Kysil, Volodymyr; Khvat, Alexander; Tsirulnikov, Sergey; Tkachenko, Sergey; Ivachtchenko, Alexandre; Gein, Vladimir L.; Panova, Olga S.; Ilyn, Alexey P.; Kravchenko, Dmitri V.; Potapov, Victor V.; Ivachtchenko, Alexandre V.; Vichegjanina, V. N.; Levandovskaya, E. B.; Gein, V. L.; Vahrin, M. I.; Vladimirov, I. N.; Zorina, A. A.; Nosova, N. V.; Gein, V. L.; Fedorova, O. V.; Vahrin, M. I.

    Multi-component and domino reactions are efficient and effective methods in the sustainable and diversity-oriented synthesis of heterocycles. In particular, transition metal-catalyzed multi-component sequences have recently gained considerable interest. Based upon the Sonogashira entry to alkynones, alkenones, and intermediate allenes, we have opened new avenues to the one-pot synthesis of numerous classes of heterocyclic frameworks in an MCR fashion. This methodological approach has now found various applications in one-pot syntheses of functional chromophores, pharmaceutically active compounds, and marine alkaloids and derivatives.

  20. Degradation of 4-chloro-3-nitrophenol via a novel intermediate, 4-chlororesorcinol by Pseudomonas sp. JHN

    NASA Astrophysics Data System (ADS)

    Arora, Pankaj Kumar; Srivastava, Alok; Singh, Vijay Pal

    2014-03-01

    A 4-chloro-3-nitrophenol (4C3NP)-mineralizing bacterium, Pseudomonas sp. JHN was isolated from a waste water sample collected from a chemically-contaminated area, India by an enrichment method. Pseudomonas sp. JHN utilized 4C3NP as a sole carbon and energy source and degraded it with the release of stoichiometric amounts of chloride and nitrite ions. Gas chromatography and gas chromatography-mass spectrometry detected 4-chlororesorcinol as a major metabolite of the 4C3NP degradation pathway. Inhibition studies using 2,2'-dipyridyl showed that 4-chlororesorcinol is a terminal aromatic compound in the degradation pathway of 4C3NP. The activity for 4C3NP-monooxygenase was detected in the crude extracts of the 4C3NP-induced JHN cells that confirmed the formation of 4-chlororesorcinol from 4C3NP. The capillary assay showed that Pseudomonas sp. JHN exhibited chemotaxis toward 4C3NP. The bioremediation capability of Pseudomonas sp. JHN was monitored to carry out the microcosm experiments using sterile and non-sterile soils spiked with 4C3NP. Strain JHN degraded 4C3NP in sterile and non-sterile soil with same degradation rates. This is the first report of (i) bacterial degradation and bioremediation of 4C3NP, (ii) formation of 4-chlororesorcinol in the degradation pathway of 4C3NP, (iii) bacterial chemotaxis toward 4C3NP.

  1. Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation.

    PubMed Central

    Newman, L M; Wackett, L P

    1997-01-01

    Trichloroethylene is oxidized by several types of nonspecific bacterial oxygenases. Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover-dependent inactivation in vivo. In this work, the oxidation of trichloroethylene was studied with purified toluene 2-monooxygenase. All three purified toluene 2-monooxygenase protein components and NADH were required to reconstitute full trichloroethylene oxidation activity in vitro. The apparent Km and Vmax were 12 microM and 37 nmol per min per mg of hydroxylase component, respectively. Ten percent of the full activity was obtained when the small-molecular-weight enzyme component was omitted. The stable oxidation products, accounting for 84% of the trichloroethylene oxidized, were carbon monoxide, formic acid, glyoxylic acid, and covalently modified oxygenase proteins that constituted 12% of the reacted [14C]trichloroethylene. The stable oxidation products may all derive from the unstable intermediate trichloroethylene epoxide that was trapped by reaction with 4-(p-nitrobenzyl)pyridine. Chloral hydrate and dichloroacetic acid were not detected. This finding differs from that with soluble methane monooxygenase and cytochrome P-450 monooxygenase, which produce chloral hydrate. Trichloroethylene-dependent inactivation of toluene 2-monooxygenase activity was observed. All of the protein components were covalently modified during the oxidation of trichloroethylene. The addition of cysteine to reaction mixtures partially protected the enzyme system against inactivation, most notably protecting the NADH-oxidoreductase component. This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase. PMID:8981984

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

    PubMed Central

    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

  3. Recent developments in asymmetric multicomponent reactions.

    PubMed

    de Graaff, Corien; Ruijter, Eelco; Orru, Romano V A

    2012-05-21

    Multicomponent reactions (MCRs) receive increasing attention because they address both diversity and complexity in organic synthesis. Thus, in principle diverse sets of relatively complex structures can be generated from simple starting materials in a single reaction step. The ever increasing need for optically pure compounds for pharmaceutical and agricultural applications as well as for catalysis promotes the development of asymmetric multicomponent reactions. In recent years, asymmetric multicomponent reactions have been applied to the total synthesis of various enantiopure natural products and commercial drugs, reducing the number of required reaction steps significantly. Although many developments in diastereoselective MCRs have been reported, the field of catalytic enantioselective MCRs has just started to blossom. This critical review describes developments in both diastereoselective and catalytic enantioselective multicomponent reactions since 2004. Significantly broadened scopes, new techniques, more environmentally benign methods and entirely novel MCRs reflect the increasingly inventive paths that synthetic chemist follow in this field. Until recently, enantioselective transition metal-catalyzed MCRs represented the majority of catalytic enantioselective MCRs. However, metal contamination is highly undesirable for drug synthesis. The emergence of organocatalysis greatly influences the quest for new asymmetric MCRs. PMID:22546840

  4. Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology

    PubMed Central

    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. PMID:26347662

  5. Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

    SciTech Connect

    Strushkevich, Natallia; MacKenzie, Farrell; Cherkesova, Tatyana; Grabovec, Irina; Usanov, Sergey; Park, Hee-Won

    2011-09-06

    In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1 - the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 {angstrom} away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

  6. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  7. Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology.

    PubMed

    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. PMID:26347662

  8. Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

    PubMed Central

    Hyman, M R; Kim, C Y; Arp, D J

    1990-01-01

    Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (less than 400 microM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazine-oxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent O2 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper. Images PMID:2118501

  9. Mechanism of Action of a Flavin-Containing Monooxygenase

    SciTech Connect

    Eswaramoorthy,S.; Bonanno, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein-cofactor and protein-substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein-cofactor and protein-substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4{alpha}-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.

  10. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  11. Effects of zinc on particulate methane monooxygenase activity and structure.

    PubMed

    Sirajuddin, Sarah; Barupala, Dulmini; Helling, Stefan; Marcus, Katrin; Stemmler, Timothy L; Rosenzweig, Amy C

    2014-08-01

    Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Zinc is a known inhibitor of pMMO, but the details of zinc binding and the mechanism of inhibition are not understood. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. The 2.6 Å resolution crystal structure of Methylocystis species strain Rockwell pMMO reveals two previously undetected bound lipids, and metal soaking experiments identify likely locations for the two zinc inhibition sites. The first is the crystallographic zinc site in the pmoC subunit, and zinc binding here leads to the ordering of 10 previously unobserved residues. A second zinc site is present on the cytoplasmic side of the pmoC subunit. Parallels between these results and zinc inhibition studies of several respiratory complexes suggest that zinc might inhibit proton transfer in pMMO. PMID:24942740

  12. Effects of Zinc on Particulate Methane Monooxygenase Activity and Structure*

    PubMed Central

    Sirajuddin, Sarah; Barupala, Dulmini; Helling, Stefan; Marcus, Katrin; Stemmler, Timothy L.; Rosenzweig, Amy C.

    2014-01-01

    Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Zinc is a known inhibitor of pMMO, but the details of zinc binding and the mechanism of inhibition are not understood. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. The 2.6 Å resolution crystal structure of Methylocystis species strain Rockwell pMMO reveals two previously undetected bound lipids, and metal soaking experiments identify likely locations for the two zinc inhibition sites. The first is the crystallographic zinc site in the pmoC subunit, and zinc binding here leads to the ordering of 10 previously unobserved residues. A second zinc site is present on the cytoplasmic side of the pmoC subunit. Parallels between these results and zinc inhibition studies of several respiratory complexes suggest that zinc might inhibit proton transfer in pMMO. PMID:24942740

  13. Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach.

    PubMed Central

    Burnet, M.; Lafontaine, P. J.; Hanson, A. D.

    1995-01-01

    The osmoprotectant glycine betaine is synthesized via the path-way choline -> betaine aldehyde -> glycine betaine. In spinach (Spinacia oleracea), the first step is catalyzed by choline monooxygenase (CMO), and the second is catalyzed by betaine aldehyde dehydrogenase. Because betaine aldehyde is unstable and not easily detected, we developed a coupled radiometric assay for CMO. [14C]Choline is used as substrate; NAD+ and betaine aldehyde dehydrogenase prepared from Escherichia coli are added to oxidize [14C]betaine aldehyde to [14C]glycine betaine, which is isolated by ion exchange. The assay was used in the purification of CMO from leaves of salinized spinach. The 10-step procedure included polyethylene glycol precipitation, polyethyleneimine precipitation, hydrophobic interaction, anion exchange on choline-Sepharose, dimethyldiethanolamine-Sepharose, and Mono Q, hydroxyapatite, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel filtration, overall purification was about 600-fold and recovery of activity was 0.5%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a polypeptide with a molecular mass of 45 kD. Taken with the value of 98 kD estimated for native CMO (R. Brouquisse, P. Weigel, D. Rhodes, C.F. Yocum, A.D. Hanson [1989] Plant Physiol 90: 322-329), this indicates that CMO is a homodimer. CMO preparations were red-brown, showed absorption maxima at 329 and 459 nm, and lost color upon dithionite addition, suggesting that CMO is an iron-sulfur protein. PMID:12228495

  14. Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration

    PubMed Central

    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

  15. Activation of enzymatic chitin degradation by a lytic polysaccharide monooxygenase.

    PubMed

    Hamre, Anne Grethe; Eide, Kristine B; Wold, Hanne H; Sørlie, Morten

    2015-04-30

    For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent k(cat) (k(cat)(app)) values on a β-chitin substrate. k(cat)(app) values were determined to be 1.7±0.1 s(-1) and 1.7±0.1 s(-1) for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s(-1) for the endo-active ChiC. The addition of CBP21 boosted the k(cat)(app) values of ChiA and ChiB giving values of 11.1±1.5 s(-1) and 13.9±1.4 s(-1), while there was no effect on ChiC (0.9±0.1 s(-1)). PMID:25812992

  16. Multicomponent dynamical nucleation theory and sensitivity analysis.

    PubMed

    Kathmann, Shawn M; Schenter, Gregory K; Garrett, Bruce C

    2004-05-15

    Vapor to liquid multicomponent nucleation is a dynamical process governed by a delicate interplay between condensation and evaporation. Since the population of the vapor phase is dominated by monomers at reasonable supersaturations, the formation of clusters is governed by monomer association and dissociation reactions. Although there is no intrinsic barrier in the interaction potential along the minimum energy path for the association process, the formation of a cluster is impeded by a free energy barrier. Dynamical nucleation theory provides a framework in which equilibrium evaporation rate constants can be calculated and the corresponding condensation rate constants determined from detailed balance. The nucleation rate can then be obtained by solving the kinetic equations. The rate constants governing the multistep kinetics of multicomponent nucleation including sensitivity analysis and the potential influence of contaminants will be presented and discussed. PMID:15267849

  17. Regulation of bacterial methane oxidation: transcription of the soluble methane mono-oxygenase operon of Methylococcus capsulatus (Bath) is repressed by copper ions.

    PubMed

    Nielsen, A K; Gerdes, K; Degn, H; Murrell, J C

    1996-05-01

    Methane is oxidized to methanol by the enzyme methane mono-oxygenase (MMO) in methanotrophic bacteria. In previous work, this multicomponent enzyme system has been extensively characterized at the biochemical and molecular level. Copper ions have been shown to irreversibly inhibit MMO activity in vivo and in vitro, but the effect of copper ions on transcription of the genes encoding the soluble (cytoplasmic) MMO (sMMO) has not previously been investigated. To examine more closely the regulation of bacterial methane oxidation and to determine the role of copper in this process, we have investigated transcriptional regulation of the sMMO gene cluster in the methanotrophic bacterium Methylococcus capsulatus (Bath). Using Northern blot analysis and primer extension experiments, it was shown that the six ORFs of the sMMO gene cluster are organized as an operon and the transcripts produced upon expression of this operon have been identified. The synthesis of these transcripts was under control of a single copper-regulated promoter, which is as yet not precisely defined. PMID:8704968

  18. Silver and gold-catalyzed multicomponent reactions

    PubMed Central

    Abbiati, Giorgio

    2014-01-01

    Summary Silver and gold salts and complexes mainly act as soft and carbophilic Lewis acids even if their use as σ-activators has been rarely reported. Recently, transformations involving Au(I)/Au(III)-redox catalytic systems have been reported in the literature. In this review we highlight all these aspects of silver and gold-mediated processes and their application in multicomponent reactions. PMID:24605168

  19. Diffusion Of Mass In Evaporating Multicomponent Drops

    NASA Technical Reports Server (NTRS)

    Bellan, Josette; Harstad, Kenneth G.

    1992-01-01

    Report summarizes study of diffusion of mass and related phenomena occurring in evaporation of dense and dilute clusters of drops of multicomponent liquids intended to represent fuels as oil, kerosene, and gasoline. Cluster represented by simplified mathematical model, including global conservation equations for entire cluster and conditions on boundary between cluster and ambient gas. Differential equations of model integrated numerically. One of series of reports by same authors discussing evaporation and combustion of sprayed liquid fuels.

  20. Multiphase, Multicomponent Compressibility in Geothermal Reservoir Engineering

    SciTech Connect

    Macias-Chapa, L.; Ramey, H.J. Jr.

    1987-01-20

    Coefficients of compressibilities below the bubble point were computer with a thermodynamic model for single and multicomponent systems. Results showed coefficients of compressibility below the bubble point larger than the gas coefficient of compressibility at the same conditions. Two-phase compressibilities computed in the conventional way are underestimated and may lead to errors in reserve estimation and well test analysis. 10 refs., 9 figs.

  1. Novel Reagents for Multi-Component Reactions

    NASA Astrophysics Data System (ADS)

    Wang, Yanguang; Basso, Andrea; Nenajdenko, Valentine G.; Gulevich, Anton V.; Krasavin, Mikhail; Bushkova, Ekaterina; Parchinsky, Vladislav; Banfi, Luca; Basso, Andrea; Cerulli, Valentina; Guanti, Giuseppe; Riva, Renata; Rozentsveig, Igor B.; Rozentsveig, Gulnur N.; Popov, Aleksandr V.; Serykh, Valeriy J.; Levkovskaya, Galina G.; Cao, Song; Shen, Li; Liu, Nianjin; Wu, Jingjing; Li, Lina; Qian, Xuhong; Chen, Xiaopeng; Wang, Hongbo; Feng, Jinwu; Wang, Yanguang; Lu, Ping; Heravi, Majid M.; Sadjadi, Samaheh; Kazemizadeh, Ali Reza; Ramazani, Ali; Kudyakova, Yulia S.; Goryaeva, Marina V.; Burgart, Yanina V.; Saloutin, Victor I.; Mossetti, Riccardo; Pirali, Tracey; Tron, Gian Cesare; Rozhkova, Yulia S.; Mayorova, Olga A.; Shklyaev, Yuriy V.; Zhdanko, Alexander G.; Nenajdenko, Valentine G.; Stryapunina, Olga G.; Plekhanova, Irina V.; Glushkov, Vladimir A.; Shklyaev, Yurii V.

    Ketenimines are a class of versatile and highly reactive intermediates that can participate in a variety of organic reactions, such as nucleophilic additions, radical additions, [2 + 2] and [2 + 4] cycloadditions, and sigmatropic rearrangements. In this presentation, we report on a series of multi-component reactions that involve a ketenimine intermediate. These reactions could furnish diverse heterocyclic compounds, including functionalized iminocoumarin, iminodihydroqunolines, iminothiochromens, pyrrolines, isoquinolines, pyridines, β-lactams, imino-1,2-dihydrocoumarins, and benzimidazoles.

  2. Predicting phase behavior in multicomponent mixtures.

    PubMed

    Jacobs, William M; Frenkel, Daan

    2013-07-14

    Mixtures with a large number of components can undergo phase transitions of a hybrid character, with both condensation and demixing contributions. We describe a robust Monte Carlo simulation method for calculating phase coexistence in multicomponent mixtures. We use this approach to study the phase behavior of lattice models of multicomponent mixtures with strongly varying pair interactions. Such a system can be thought of as a simplified model of the cytosol, with both specific and nonspecific interactions. We show that mapping a multicomponent mixture onto an approximately equivalent one-component system yields both upper and lower bounds on the maximum solute volume fraction of a stable, homogeneous phase. By following the minimum excess-free-energy path from the dilute phase free-energy minimum, we predict the difference in composition between the condensed and dilute phases at the boundary of the homogeneous phase. We find that this "direction" of phase separation rarely aligns with the dominant direction of density fluctuations in the dilute phase. We also show that demixing transitions tend to lower the maximum solute volume fraction at which the homogeneous phase is stable. By considering statistical ensembles of mixtures with random interactions, we show that the demixing contribution to phase separation is self-averaging and dependent only on the mean and variance of the distribution of interactions. PMID:23862930

  3. Multicomponent gas sorption Joule-Thomson refrigeration

    NASA Technical Reports Server (NTRS)

    Jones, Jack A. (Inventor); Petrick, S. Walter (Inventor); Bard, Steven (Inventor)

    1991-01-01

    The present invention relates to a cryogenic Joule-Thomson refrigeration capable of pumping multicomponent gases with a single stage sorption compressor system. Alternative methods of pumping a multicomponent gas with a single stage compressor are disclosed. In a first embodiment, the sorbent geometry is such that a void is defined near the output of the sorption compressor. When the sorbent is cooled, the sorbent primarily adsorbs the higher boiling point gas such that the lower boiling point gas passes through the sorbent to occupy the void. When the sorbent is heated, the higher boiling point gas is desorbed at high temperature and pressure and thereafter propels the lower boiling point gas out of the sorption compressor. A mixing chamber is provided to remix the constituent gases prior to expansion of the gas through a Joule-Thomson valve. Other methods of pumping a multicomponent gas are disclosed. For example, where the sorbent is porous and the low boiling point gas does not adsorb very well, the pores of the sorbent will act as a void space for the lower boiling point gas. Alternatively, a mixed sorbent may be used where a first sorbent component physically adsorbs the high boiling point gas and where the second sorbent component chemically absorbs the low boiling point gas.

  4. Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria

    SciTech Connect

    Jahng, D.; Kim, C.S.; Wood, T.K.; Hanson, R.S.

    1996-08-05

    By complementing cell-free extracts of Pseudomonas putida F1/pSMMO20 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichospoirium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMMO20 and pSMMO50 into various bacterial species including pseudomonads and {alpha}-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO20 (resulting in pSMMO40) and found to enhance plasmid stability in P. putida F1 and R. meliloti 102F34 (first report of hok/sok in Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(II), Ni(II), and Zn(II)] and chloramphenicol from the medium and by supplementing with an iron source (3.6 {micro}M of ferrous ammonium sulfate). Using chemostat-grown P. putida F1/pSMMO40, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity.

  5. Transcriptional Regulation of the Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression in Response to Xylella fastidiosa Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are a group of versatile redox proteins that mediate the biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds which act as plant defense agents. To determine if cytochrome P450 monooxygenases are involved in defense response to...

  6. Selective Usage of Transcription Initiation and Polyadenylation Sites in Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  7. THE BIOCHEMICAL CHARACTERIZATION OF FERRET CAROTENE-9', 10'-MONOOXYGENASE CATALYZING CLEAVAGE OF CAROTENOIDS IN VITRO AND IN VIVO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that beta -carotene 15,15'-monooxygenase (CMO1) catalyzes the cleavage of beta -carotene at the central carbon 15, 15’-double bond, but cleaves lycopene with much lower activity. However, expressing the mouse carotene-9’,10’-monooxygenase (CMO2) in beta-carotene/lycopene...

  8. A family of starch-active polysaccharide monooxygenases

    PubMed Central

    Vu, Van V.; Beeson, William T.; Span, Elise A.; Farquhar, Erik R.; Marletta, Michael A.

    2014-01-01

    The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries. PMID:25201969

  9. A family of starch-active polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Beeson, William T; Span, Elise A; Farquhar, Erik R; Marletta, Michael A

    2014-09-23

    The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries. PMID:25201969

  10. Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

    PubMed

    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. PMID:26862199

  11. Terpene hydroxylation with microbial cytochrome P450 monooxygenases.

    PubMed

    Janocha, Simon; Schmitz, Daniela; Bernhardt, Rita

    2015-01-01

    Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes. PMID:25682070

  12. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Zahn, J A; DiSpirito, A A

    1996-01-01

    An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide. PMID:8576034

  13. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Zahn, J A; DiSpirito, A A

    1996-02-01

    An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide. PMID:8576034

  14. Metabolic pathway involved in 2-methyl-6-ethylaniline degradation by Sphingobium sp. strain MEA3-1 and cloning of the novel flavin-dependent monooxygenase system meaBA.

    PubMed

    Dong, Weiliang; Chen, Qiongzhen; Hou, Ying; Li, Shuhuan; Zhuang, Kai; Huang, Fei; Zhou, Jie; Li, Zhoukun; Wang, Jue; Fu, Lei; Zhang, Zhengguang; Huang, Yan; Wang, Fei; Cui, Zhongli

    2015-12-01

    2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02' in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase. PMID:26386060

  15. Metabolic Pathway Involved in 2-Methyl-6-Ethylaniline Degradation by Sphingobium sp. Strain MEA3-1 and Cloning of the Novel Flavin-Dependent Monooxygenase System meaBA

    PubMed Central

    Dong, Weiliang; Chen, Qiongzhen; Hou, Ying; Li, Shuhuan; Zhuang, Kai; Huang, Fei; Zhou, Jie; Li, Zhoukun; Wang, Jue; Fu, Lei; Zhang, Zhengguang; Huang, Yan; Wang, Fei

    2015-01-01

    2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02′ in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase. PMID:26386060

  16. A study of the hepatic microsomal monooxygenase of sea birds and its relationship to organochlorine pollutants.

    PubMed

    Knight, G C; Walker, C H

    1982-01-01

    1. The levels of hepatic microsomal monooxygenase in sea birds were determined using organochlorine substrates. Levels of cytochrome P450 and organochlorine residues were also measured. 2. The razorbill (Alca torda) and puffin (Fratercula arctica) showed highly variable activities which were resolved into multiple peaks on frequency diagrams. 3. The most active individuals amongst razorbills were early season females with large ovaries. 4. The properties of monooxygenase from individuals of low and high activity were compared. 5. The results are discussed in relation to PCB pollution. PMID:6128175

  17. Toluene 2-Monooxygenase-Dependent Growth of Burkholderia cepacia G4/PR1 on Diethyl Ether

    PubMed Central

    Hur, H.; Newman, L. M.; Wackett, L. P.; Sadowsky, M. J.

    1997-01-01

    Aerobic bacterial growth on aromatic hydrocarbons typically requires oxygenase enzymes, which are known to fortuitously oxidize nongrowth substrates. In this study, we found that oxidation of diethyl ether by toluene 2-monooxygenase supported more rapid growth of Burkholderia cepacia G4/PR1 than did the aromatic substrates n-propylbenzene and o-xylene. The wild-type Burkholderia cepacia G4 failed to grow on diethyl ether. Purified toluene 2-monooxygenase protein components oxidized diethyl ether stoichiometrically to ethanol and acetaldehyde. Butyl methyl ether, diethyl sulfide, and 2-chloroethyl ethyl ether were oxidized by B. cepacia G4/PR1. PMID:16535583

  18. Intermediate P* from Soluble Methane Monooxygenase Contains a Diferrous Cluster

    PubMed Central

    Banerjee, Rahul; Meier, Katlyn K.; Münck, Eckard; Lipscomb, John D.

    2013-01-01

    During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the FeIIFeII state (Hred). O2 binds rapidly at a site away from the cluster to form the FeIIFeII intermediate O, which converts to an FeIIIFeIII-peroxo intermediate P and finally to the FeIVFeIV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of Hred and O is lost. This was interpreted as signaling oxidation of the cluster, but low accumulation of P* prevented further characterization. In this study, three methods to directly detect and trap P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of Hred and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the FeIIFeII state. Thus, the loss of the g = 16 EPR results from a change of

  19. Regulation of Hydroxylation and Nitroreduction Pathways during Metabolism of the Neonicotinoid Insecticide Imidacloprid by Pseudomonas putida.

    PubMed

    Lu, Tian-Qi; Mao, Shi-Yun; Sun, Shi-Lei; Yang, Wen-Long; Ge, Feng; Dai, Yi-Jun

    2016-06-22

    Imidacloprid (IMI) is mainly metabolized via nitroreduction and hydroxylation pathways, which produce different metabolites that are toxic to mammals and insects. However, regulation of IMI metabolic flux between nitroreduction and hydroxylation pathways is still unclear. In this study, Pseudomonas putida was found to metabolize IMI to 5-hydroxy and nitroso IMI and was therefore used for investigating the regulation of IMI metabolic flux. The cell growth time, cosubstrate, dissolved oxygen concentration, and pH showed significant effect on IMI degradation and nitroso and 5-hydroxy IMI formation. Gene cloning and overexpression in Escherichia coli proved that P. putida KT2440 aldehyde oxidase mediated IMI nitroreduction to nitroso IMI, while cytochrome P450 monooxygenase (CYP) failed to improve IMI hydroxylation. Moreover, E. coli cells without CYP could hydroxylate IMI, demonstrating the role of a non-CYP enzyme in IMI hydroxylation. Thus, the present study helps to further understand the environmental fate of IMI and its underlying mechanism. PMID:27230024

  20. Simulation of paraequilibrium growth in multicomponent systems

    NASA Astrophysics Data System (ADS)

    Ghosh, G.; Olson, G. B.

    2001-03-01

    A methodology to simulate paraequilibrium (PE) growth in multicomponent systems using the DIC-TRA (Diffusion-Controlled Transformation) software is presented. For any given multicomponent system containing substitutional and interstitial elements, the basic approach is to define a hypothetical element Z, whose thermodynamic and mobility parameters are expressed in terms of the weighted average (with respect to site fraction) of the thermodynamic parameters and mobilities of the substitutional alloying elements. This procedure facilitates the calculation of PE phase diagrams and the PE growth simulations directly in the Thermo-Calc and DICTRA software, respectively. The results of two distinct case studies in multicomponent alloys are presented. In the first example, we simulate the isothermal growth of PE cementite in an Fe-C-Co-Cr-Mo-Ni secondary hardening steel during tempering. This is of practical importance in modeling the carbide precipitation kinetics during secondary hardening. In the second example, we have presented the results of PE ferrite growth during continuous cooling from an intercritical temperature in an Fe-Al-C-Mn-Si low-alloy steel. This is of importance to the design of triple-phase steels containing an austenite that has optimum stability, to facilitate stress-induced transformation under dynamic loading. The results of both simulations are in good accord with experimental results. The model calculations do not consider any resistive or dissipative forces, such as the interfacial energy, strain energy, or solute drag, and, as a result, the interface velocities represent an upper limit under the available chemical driving force.

  1. Hybrid Multicomponent Hydrogels for Tissue Engineering

    PubMed Central

    Jia, Xinqiao; Kiick, Kristi L.

    2009-01-01

    Artificial ECMs that not only closely mimic the hybrid nature of the natural ECM but also provide tunable material properties and enhanced biological functions are attractive candidates for tissue engineering applications. This review summarizes recent advances in developing multicomponent hybrid hydrogels by integrating modular and heterogeneous building blocks into well-defined, multifunctional hydrogel composites. The individual building blocks can be chemically, morphologically, and functionally diverse, and the hybridization can occur at molecular level or microscopic scale. The modular nature of the designs, combined with the potential synergistic effects of the hybrid systems, has resulted in novel hydrogel matrices with robust structure and defined functions. PMID:19107720

  2. Regularized Grad equations for multicomponent plasmas

    NASA Astrophysics Data System (ADS)

    Magin, Thierry E.; Martins, Gérald; Torrilhon, Manuel

    2011-05-01

    The moment method of Grad is used to derive macroscopic conservation equations for multicomponent plasmas for small and moderate Knudsen numbers, accounting for the electromagnetic field influence and thermal nonequilibrium. In the low Knudsen number limit, the equations derived are fully consistent with those obtained by means of the Chapman-Enskog method. In particular, we have retieved the Kolesnikov effect coupling electrons and heavy particles in the case of the Boltzmann moment systems. Finally, a regularization procedure is proposed to achieve continuous shock structures at all Mach numbers.

  3. Statistical Model of Evaporating Multicomponent Fuel Drops

    NASA Technical Reports Server (NTRS)

    Harstad, Kenneth; LeClercq, Patrick; Bellan, Josette

    2007-01-01

    An improved statistical model has been developed to describe the chemical composition of an evaporating multicomponent- liquid drop and of the mixture of gases surrounding the drop. The model is intended for use in computational simulations of the evaporation and combustion of sprayed liquid fuels, which are typically mixtures of as many as hundreds of different hydrocarbon compounds. The present statistical model is an approximation designed to afford results that are accurate enough to contribute to understanding of the simulated physical and chemical phenomena, without imposing an unduly large computational burden.

  4. General Model for Multicomponent Ablation Thermochemistry

    NASA Technical Reports Server (NTRS)

    Milos, Frank S.; Marschall, Jochen; Rasky, Daniel J. (Technical Monitor)

    1994-01-01

    A previous paper (AIAA 94-2042) presented equations and numerical procedures for modeling the thermochemical ablation and pyrolysis of thermal protection materials which contain multiple surface species. This work describes modifications and enhancements to the Multicomponent Ablation Thermochemistry (MAT) theory and code for application to the general case which includes surface area constraints, rate limited surface reactions, and non-thermochemical mass loss (failure). Detailed results and comparisons with data are presented for the Shuttle Orbiter reinforced carbon-carbon oxidation protection system which contains a mixture of sodium silicate (Na2SiO3), silica (SiO2), silicon carbide (SiC), and carbon (C).

  5. Multicomponent glass fiber optic integrated structures

    NASA Astrophysics Data System (ADS)

    Pysz, Dariusz; Kujawa, Ireneusz; Szarniak, Przemyslaw; Franczyk, Marcin; Stepien, Ryszard; Buczynski, Ryszard

    2005-09-01

    A range of integrated fiber optic structures - lightguides, image guides, multicapillary arrays, microstructured (photonic) fibers - manufactured in the Institute of Electronic Materials Technology (ITME) is described. All these structures are made of multicomponent glasses (a part of them melted in ITME). They can be manufactured in similar multistep process that involves drawing glass or lightguide rods and tubes preparing glass performs, stacking a bundle with rods and (or) tubes, drawing multifiber or multicapillary performs. Structure formation, technological process, characterization and applications of different integrated structures are presented.

  6. Corrections to scaling in multicomponent polymer solutions.

    PubMed

    Pelissetto, Andrea; Vicari, Ettore

    2006-05-01

    We calculate the correction-to-scaling exponent omegaT that characterizes the approach to the scaling limit in multicomponent polymer solutions. A direct Monte Carlo determination of omegaT in a system of interacting self-avoiding walks gives omegaT=0.415+/-0.020. A field-theory analysis based on five- and six-loop perturbative series leads to omegaT=0.41+/-0.04. We also verify the renormalization-group predictions for the scaling behavior close to the ideal-mixing point. PMID:16802958

  7. [Meningoencephalitis caused by Pseudomonas cepacia].

    PubMed

    Pérez Monrás, Miriam Fina; Batlle Almodóvar, María del Carmen; González, Cernero; Tamargo Martínez, Isis; Meneses, Félix Dickinson

    2006-01-01

    A case of meningoencephalitis of bacterial etiology caused by Pseudomonas cepacia was described. The strain was received at the Reference Laboratory of Bacterial Acute Respiratory Infections of "Pedro Kouri" Institute of Tropical Medicine, where its microbiological identification was confirmed. This isolation was a finding in an adult immunocompetent patient. The evolution was favourable with no sequelae for his future life. Pseudomona cepacia has been associated with respiratory infections in patients with cystic fibrosis. Patients with Pseudomonas cepacia may be asymptomatic or present fatal acute and fulminant infection. PMID:23427437

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

    SciTech Connect

    Carlin, DA; Bertolani, SJ; Siegel, JB

    2015-01-01

    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.

  9. A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

    PubMed

    Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

    2012-06-01

    N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. PMID:22410281

  10. Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications

    PubMed Central

    Ceccoli, Romina D.; Bianchi, Dario A.; Rial, Daniela V.

    2014-01-01

    External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years. PMID:24567729

  11. Cloning, expression and characterization of a eukaryotic cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011.

    PubMed

    Leipold, Friedemann; Wardenga, Rainer; Bornscheuer, Uwe T

    2012-05-01

    In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer-Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme. PMID:22075635

  12. Expression of Individual Copies of Methylococcus capsulatus Bath Particulate Methane Monooxygenase Genes

    PubMed Central

    Stolyar, Sergei; Franke, Marion; Lidstrom, Mary E.

    2001-01-01

    The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth. PMID:11160118

  13. Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes.

    PubMed

    Stolyar, S; Franke, M; Lidstrom, M E

    2001-03-01

    The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth. PMID:11160118

  14. Identification of Cytochrome P450 monooxygenases in Diaphornia citri, an economically important psyllid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty P450’s in the CYP monooxygenases were identified in the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The psyllid is responsible for the transmission of Candidatus Liberibacter asiaticus, the causative agent of huanglongbing ( HLB), also known as citrus greening dise...

  15. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  16. Biology of Pseudomonas stutzeri

    PubMed Central

    Lalucat, Jorge; Bennasar, Antoni; Bosch, Rafael; García-Valdés, Elena; Palleroni, Norberto J.

    2006-01-01

    Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri. PMID:16760312

  17. Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600.

    PubMed Central

    Knobel, H R; Egli, T; van der Meer, J R

    1996-01-01

    A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression. PMID:8892809

  18. Steady-state kinetics and spectroscopic characterization of enzyme-tRNA interactions for the non-heme diiron tRNA-monooxygenase, MiaE.

    PubMed

    Subedi, Bishnu P; Corder, Andra L; Zhang, Siai; Foss, Frank W; Pierce, Brad S

    2015-01-20

    MiaE [2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA monooxygenase] isolated from Salmonella typhimurium is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide (ms(2)i(6)A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N(6)-(4-hydroxyisopentenyl)-adenosine(37). In this work, isopentenylated tRNA substrates for MiaE were produced from small RNA oligomers corresponding to the anticodon stem loop (ACSL) region of tRNA(Trp) using recombinant MiaA and dimethylallyl pyrophosphate. Steady-state rates for MiaE-catalyzed substrate hydroxylation were determined using recombinant ferredoxin (Fd) and ferredoxin reductase (FdR) to provide a catalytic electron transport chain (ETC) using NADPH as the sole electron source. As with previously reported peroxide-shunt assays, steady-state product formation retains nearly stoichiometric (>98%) E stereoselectivity. MiaE-catalyzed i(6)A-ACSL(Trp) hydroxylation follows Michaelis-Menten saturation kinetics with kcat, KM, and V/K determined to be 0.10 ± 0.01 s(-1), 9.1 ± 1.5 μM, and ∼11000 M(-1) s(-1), respectively. While vastly slower, MiaE-catalyzed hydroxylation of free i(6)A nucleoside could also be observed using the (Fd/FdR)-ETC assay. By comparison to the V/K determined for i(6)A-ACSL substrates, an ∼6000-fold increase in enzymatic efficiency is imparted by ACSL(Trp)-MiaE interactions. The impact of substrate tRNA-MiaE interactions on protein secondary structure and active site electronic configuration was investigated using circular dichroism, dual-mode X-band electron paramagnetic resonance, and Mössbauer spectroscopies. These studies demonstrate that binding of tRNA to MiaE induces a protein conformational change that influences the electronic structure of the diiron site analogous to what has been observed for various bacterial multicomponent diiron monooxygenases upon titration with their corresponding effector

  19. Lattice Boltzmann algorithm for continuum multicomponent flow.

    PubMed

    Halliday, I; Hollis, A P; Care, C M

    2007-08-01

    We present a multicomponent lattice Boltzmann simulation for continuum fluid mechanics, paying particular attention to the component segregation part of the underlying algorithm. In the principal result of this paper, the dynamics of a component index, or phase field, is obtained for a segregation method after U. D'Ortona [Phys. Rev. E 51, 3718 (1995)], due to Latva-Kokko and Rothman [Phys. Rev. E 71 056702 (2005)]. The said dynamics accord with a simulation designed to address multicomponent flow in the continuum approximation and underwrite improved simulation performance in two main ways: (i) by reducing the interfacial microcurrent activity considerably and (ii) by facilitating simulational access to regimes of flow with a low capillary number and drop Reynolds number [I. Halliday, R. Law, C. M. Care, and A. Hollis, Phys. Rev. E 73, 056708 (2006)]. The component segregation method studied, used in conjunction with Lishchuk's method [S. V. Lishchuk, C. M. Care, and I. Halliday, Phys. Rev. E 67, 036701 (2003)], produces an interface, which is distributed in terms of its component index; however, the hydrodynamic boundary conditions which emerge are shown to support the notion of a sharp, unstructured, continuum interface. PMID:17930175

  20. Theory of margination in confined multicomponent suspensions

    NASA Astrophysics Data System (ADS)

    Henriquez Rivera, Rafael; Sinha, Kushal; Graham, Michael

    2015-11-01

    In blood flow, leukocytes and platelets tend to segregate near the vessel walls; this is known as margination. Margination of leukocytes and platelets is important in physiological processes, medical diagnostics and drug delivery. A mechanistic theory is developed to describe flow-induced segregation in confined multicomponent suspensions of deformable particles such as blood. The theory captures the essential features of margination by describing it in terms of two key competing processes in these systems at low Reynolds number: wall-induced migration and hydrodynamic pair collisions. The theory also includes the effect of physical properties of the deformable particles and molecular diffusion. Several regimes of segregation are identified, depending on the value of a ``margination parameter'' M. Moreover, there is a critical value of M below which a sharp ``drainage transition'' occurs: one component is completely depleted from the bulk flow to the vicinity of the walls. Direct hydrodynamic simulations also display this transition in suspensions where the components differ in size or flexibility. The developed mechanistic theory leads to substantial insight into the origins of margination and will help in guiding development of new technologies involving multicomponent suspensions. This work was supported by NSF grant CBET-1436082.

  1. Pseudomonas aeruginosa in Healthcare Settings

    MedlinePlus

    ... becoming more difficult to treat because of increasing antibiotic resistance. Selecting the right antibiotic usually requires that a ... to help educate people about Pseudomonas infections, and antibiotic resistance, and to encourage prevention activities and healthy behaviors ...

  2. Structures of the Apo and FAD-Bound Forms of 2-Hydroxybiphenyl 3-monooxygenase (HbpA) Locate Activity Hotspots Identified by Using Directed Evolution

    PubMed Central

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

    2015-01-01

    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. PMID:25737306

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

    PubMed

    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. PMID:25737306

  4. Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms.

    PubMed

    Karande, Rohan; Debor, Linde; Salamanca, Diego; Bogdahn, Fabian; Engesser, Karl-Heinrich; Buehler, Katja; Schmid, Andreas

    2016-01-01

    The applications of biocatalysts in chemical industries are characterized by activity, selectivity, and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp. CHX100 and their functional expression in recombinant Pseudomonas taiwanensis VLB120 enabled efficient oxidation of cyclohexane to cyclohexanol. Although initial resting cell activities of 20 U gCDW (-1) were achieved, the rapid decrease in catalytic activity due to the toxicity of cyclohexane prevented synthetic applications. Cyclohexane toxicity was reduced and cellular activities stabilized over the reaction time by delivering the toxic substrate through the vapor phase and by balancing the aqueous phase mass transfer with the cellular conversion rate. The potential of this novel CYP enzyme was exploited by transferring the shake flask reaction to an aqueous-air segmented flow biofilm membrane reactor for maximizing productivity. Cyclohexane was continuously delivered via the silicone membrane. This ensured lower reactant toxicity and continuous product formation at an average volumetric productivity of 0.4 g L tube (-1) h(-1) for several days. This highlights the potential of combining a powerful catalyst with a beneficial reactor design to overcome critical issues of cyclohexane oxidation to cyclohexanol. It opens new opportunities for biocatalytic transformations of compounds which are toxic, volatile, and have low solubility in water. PMID:26153144

  5. Cloning of Toluene 4-Monooxygenase Genes and Application of Two-Phase System to the Production of the Anticancer Agent, Indirubin.

    PubMed

    Wongsaroj, Lampet; Sallabhan, Ratiboot; Dubbs, James M; Mongkolsuk, Skorn; Loprasert, Suvit

    2015-08-01

    Indirubin is a strong inhibitor of several eukaryotic cell signaling pathways and shows promise as a treatment for myelocytic leukemia and Alzheimer's disease. The tmoABCDEF operon, encoding the components of a novel toluene 4-monooxygenase from the paint factory soil isolate, Pseudomonas sp. M4, was cloned and expressed in Escherichia coli. E. coli::pKSR12 expressing the tmo genes was used to develop a two-phase [dioctyl phthalate (DOP)/aqueous medium] culture system that was optimized to obtain maximal yields of indirubin from the starting substrate, indole. DOP was used as the organic phase to solubilize and sequester the toxic indole substrate, making possible the use of high indole concentrations that would otherwise interfere with growth in aqueous media. A 50 % (v/v) DOP two-phase system using tryptophan medium containing 3 mM cysteine, 5 mM indole, and 1 mM isatin yielded 102.4 mg/L of indirubin with no conversion of indole to the less valuable alternate product, indigo. PMID:25779640

  6. Chloroform Cometabolism by Butane-Grown CF8, Pseudomonas butanovora, and Mycobacterium vaccae JOB5 and Methane-Grown Methylosinus trichosporium OB3b

    PubMed Central

    Hamamura, N.; Page, C.; Long, T.; Semprini, L.; Arp, D. J.

    1997-01-01

    Chloroform (CF) degradation by a butane-grown enrichment culture, CF8, was compared to that by butane-grown Pseudomonas butanovora and Mycobacterium vaccae JOB5 and to that by a known CF degrader, Methylosinus trichosporium OB3b. All three butane-grown bacteria were able to degrade CF at rates comparable to that of M. trichosporium. CF degradation by all four bacteria required O(inf2). Butane inhibited CF degradation by the butane-grown bacteria, suggesting that butane monooxygenase is responsible for CF degradation. P. butanovora required exogenous reductant to degrade CF, while CF8 and M. vaccae utilized endogenous reductants. Prolonged incubation with CF resulted in decreased CF degradation. CF8 and P. butanovora were more sensitive to CF than either M. trichosporium or M. vaccae. CF degradation by all three butane-grown bacteria was inactivated by acetylene, which is a mechanism-based inhibitor for several monooxygenases. Butane protected all three butane-grown bacteria from inactivation by acetylene, which indicates that the same monooxygenase is responsible for both CF and butane oxidation. CF8 and P. butanovora were able to degrade other chlorinated hydrocarbons, including trichloroethylene, 1,2-cis-dichloroethylene, and vinyl chloride. In addition, CF8 degraded 1,1,2-trichloroethane. The results indicate the potential of butane-grown bacteria for chlorinated hydrocarbon transformation. PMID:16535693

  7. Gauge transformation and symmetries of the commutative multicomponent BKP hierarchy

    NASA Astrophysics Data System (ADS)

    Li, Chuanzhong

    2016-01-01

    In this paper, we defined a new multi-component B type Kadomtsev-Petviashvili (BKP) hierarchy that takes values in a commutative subalgebra of {gl}(N,{{C}}). After this, we give the gauge transformation of this commutative multicomponent BKP (CMBKP) hierarchy. Meanwhile, we construct a new constrained CMBKP hierarchy that contains some new integrable systems, including coupled KdV equations under a certain reduction. After this, the quantum torus symmetry and quantum torus constraint on the tau function of the commutative multi-component BKP hierarchy will be constructed.

  8. Multicomponent processing of channel waves for anisotropy

    SciTech Connect

    Macbeth, C.; Liu, E.; Horne, S.

    1994-12-31

    Combining information derived from mode polarization and dispersion may help to limit inherent non-uniquenesses when interpreting channel wave data in anisotropic waveguides. However, estimates of these attributes require careful processing and cannot be fully understood by direct inspection of the time-series. A multicomponent analysis method is developed to separate these frequency-dependent attributes when data containing no more than a few overlapping modes is excited by several source directions. The method, a complex similarity transformation,may be viewed as the channel wave equivalent of real rotation algorithms currently employed for use with body waves. The analysis is illustrated by application to field data from an in-seam seismic survey in the U.K. displaying a dominant second generalized mode (Love-type motion). A favorable comparison is made between the observed and synthetic eigenmode dispersion, and the polarization ellipticity, sense of rotation and orientation.

  9. Critical dynamics in multicomponent lipid membranes.

    PubMed

    Haataja, Mikko

    2009-08-01

    The formation and dynamics of spatially extended compositional domains in multicomponent lipid membranes both in vivo and in vitro lie at the heart of many important biological and biophysical phenomena. While the thermodynamic basis for domain formation has been explored extensively in the past, the roles of membrane and exterior fluid hydrodynamics on domain formation kinetics have received less attention. A case in point is the impact of hydrodynamics on the dynamics of compositional heterogeneities in lipid membranes in the vicinity of a critical point. In this Rapid Communication it is argued that the asymptotic dynamic behavior of a lipid membrane system in the vicinity of a critical point is strongly influenced by hydrodynamic interactions. More specifically, a mode-coupling argument is developed which predicts a scaling behavior of lipid transport coefficients near the critical point for both symmetric and asymmetric bilayers immersed in a bulk fluid. PMID:19792068

  10. Multicomponent diffusion—A brief review

    NASA Astrophysics Data System (ADS)

    Bird, R. Byron; Klingenberg, Daniel J.

    2013-12-01

    After a brief summary of previous work on multicomponent diffusion theory, we review how the thermodynamics of irreversible processes leads us to an expression for the generalized driving force for diffusion. We then give the generalized Fick's law expression containing the diffusivities D; then we show how this may be turned "wrong-side out" using Merk's method to give the generalized Maxwell-Stefan equations containing the diffusivities Ð12≡x1x2/C12. Finally, we show how the latter are related to the diffusivities D12 that are usually reported in the literature. All results are applicable to gases or liquids. This review is restricted to presenting the basic theory in a consistent notation, and not to the applications of the theory.

  11. Determination of Stability from Multicomponent Pesticide Mixes.

    PubMed

    Dorweiler, Kelly J; Gurav, Jagdish N; Walbridge, James S; Ghatge, Vishwas S; Savant, Rahul H

    2016-08-10

    A study was conducted to evaluate the stability of 528 pesticides, metabolites, and contaminants prepared in large multicomponent mixes to enhance laboratory efficiency by allowing maximum use of the useful shelf life of the mixtures. Accelerated aging at 50 °C simulated 6 month, 1 year, and 2 year storage periods at -20 °C. Initial mixture composition was based on the instrument of analysis. After preliminary stability data had been obtained, mixtures were reformulated and re-evaluated. In all, 344 compounds showed satisfactory stability across all treatment groups, 100 compounds showed statistically significant changes between the control and the 6 month simulated storage period (27 with losses >20%), and the remainder showed borderline stability or were tested in one protocol. Stability behavior for organophosphates agreed with the proposed reaction mechanism responsible for acetylcholinesterase inhibition. A small number of compounds increased in response over time, suggesting the occurrence of degradation of precursor pesticides into these respective compounds. PMID:26937779

  12. Design of Multi-Component Reactions

    NASA Astrophysics Data System (ADS)

    Zhu, Jieping; Kaïm, Laurent El; Tron, Gian Cesare; Lavilla, Rodolfo; Banfi, Luca; Basso, Andrea; Cerulli, Valentina; Guanti, Giuseppe; Lecinska, Paulina; Riva, Renata; Arévalo, M. J.; Kielland, N.; Masdeu, C.; Miguel, M.; Isambert, N.; Lavilla, R.; Medvedeva, Alevtina S.; Novokshonov, Vladimir V.; Novokshonova, Irina A.; Demina, Maria M.; Kon'kova, Tatyana V.; Shklyaev, Yurii V.; Rozhkova, Yulia S.; Vshivkova, Tatiana S.; Stryapunina, Olga G.; Glushkov, Vladimir A.; Kharitonova, Anastasia V.; Fisyuk, Alexander S.; Mukanov, Aleksey Y.; Poendaev, Nicolay V.; Gulevich, Anton V.; Nenajdenko, Valentine G.; Ivantsova, Maria N.; Tokareva, Maria I.; Mironov, Maxim A.; Mokrushin, Vladimir S.; Pirali, Tracey; Tron, Gian Cesare; Zhu, Jieping; Rozentsveig, Igor B.; Popov, Aleksandr V.; Levkovskaya, Galina G.; Chernyshev, Kirill A.; Krivdin, Leonid B.; Tomilov, Yury V.; Platonov, Dmitry N.; Rulev, Alexander Y.; Ushakov, Igor A.; Vorobyeva, Alexandra; Ilyin, Alexey; Kysil, Volodimir; Ivachtchenko, Alexandre

    Multi-component reactions (MCRs) have now been well established as a powerful synthetic tool for creating molecular complexity and diversity and are undoubtedly well suited for the drug discovery program. Another potential that has probably received less attention among synthetic chemists is the opportunity offered by MCRs for the development of new fundamentally important transformations (reactions). Indeed, although an MCR is composed of a series of known bimolecular reactions, the overall transformation could be novel. Consequently, it provides chemists the opportunities to uncover transformations that were otherwise difficult to realize. In this talk, we will present our recent work in this field, including: (1) the oxidative homologation of aldehydes to amides, (2) the oxidative coupling of aldehydes and isocyanides to α-ketoamides, (3) oxidative isocyanide-based MCRs, and (4) the enantioselective Passerini reaction.

  13. Chemotaxis by Pseudomonas aeruginosa.

    PubMed Central

    Moulton, R C; Montie, T C

    1979-01-01

    Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa. PMID:104961

  14. Simulations of Evaporating Multicomponent Fuel Drops

    NASA Technical Reports Server (NTRS)

    Bellan, Josette; Le Clercq, Patrick

    2005-01-01

    A paper presents additional information on the subject matter of Model of Mixing Layer With Multicomponent Evaporating Drops (NPO-30505), NASA Tech Briefs, Vol. 28, No. 3 (March 2004), page 55. To recapitulate: A mathematical model of a three-dimensional mixing layer laden with evaporating fuel drops composed of many chemical species has been derived. The model is used to perform direct numerical simulations in continuing studies directed toward understanding the behaviors of sprays of liquid petroleum fuels in furnaces, industrial combustors, and engines. The model includes governing equations formulated in an Eulerian and a Lagrangian reference frame for the gas and drops, respectively, and incorporates a concept of continuous thermodynamics, according to which the chemical composition of a fuel is described by use of a distribution function. In this investigation, the distribution function depends solely on the species molar weight. The present paper reiterates the description of the model and discusses further in-depth analysis of the previous results as well as results of additional numerical simulations assessing the effect of the mass loading. The paper reiterates the conclusions reported in the cited previous article, and states some new conclusions. Some new conclusions are: 1. The slower evaporation and the evaporation/ condensation process for multicomponent-fuel drops resulted in a reduced drop-size polydispersity compared to their single-component counterpart. 2. The inhomogeneity in the spatial distribution of the species in the layer increases with the initial mass loading. 3. As evaporation becomes faster, the assumed invariant form of the molecular- weight distribution during evaporation becomes inaccurate.

  15. Characterization and Optimization Multiscale and Multicomponent Nanosystems

    NASA Astrophysics Data System (ADS)

    Nash, Kelly

    2012-10-01

    Materials with new combinations of properties are increasingly needed to meet the requirements of energy, transportation, and medical applications. The use of multi-component systems, with potentially complementary properties, represent a unique path to improve materials properties for a variety of applications. Among the most interesting applications of these materials is in the development of contrast agents in biological imaging and dynamic sensing applications. Although a variety of techniques to characterize these materials exist, noninvasive characterization methods, such as optical-based techniques, are ideal for studying these materials in their native states and for monitoring dynamic changes. The proposition becomes even more attractive when at least one of the components carries an optical signature.The use of optoacoustic (OA) is an emerging technology based on studying optically absorbing nano and microstructures in the sample by recording transit pressure waves generated from laser-induced thermal expansion. More recently OA has been developed as a vibrant technology for medical applications and some growing applications is for material characterization in research and industrial applications. Specifically, OA can assist in the characterization and optimization of composite materials containing nanoparticles when paired with other characterization techniques. The present work illustrates an overview of select hybrid nanomaterials, including their unique optoacoustic signatures utilizing an all optical OA technique. The results of this work show that optical based techniques such as OA, provide a noninvasive, nondestructive means to study multi-material, multi-scale, multi-functional materials are important in the development of novel multi-component nanomaterial schemes and elucidating the structure-function relationship in these materials.

  16. Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments.

    PubMed

    McDonald, Ian R; Miguez, Carlos B; Rogge, Gerlinde; Bourque, Denis; Wendlandt, Karin D; Groleau, Denis; Murrell, J Colin

    2006-02-01

    Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function. PMID:16448499

  17. Modelling of micro- and macrosegregation for industrial multicomponent aluminium alloys

    NASA Astrophysics Data System (ADS)

    Ellingsen, K.; Mortensen, D.; M'Hamdi, M.

    2015-06-01

    Realistic predictions of macrosegregation formation during casting of aluminium alloys requires an accurate modeling of solute microsegregation accounting for multicomponent phase diagrams and secondary phase formation. In the present work, the stand alone Alstruc model, a microsegregation model for industrial multicomponent aluminium alloys, is coupled with the continuum model ALSIM which calculates the macroscopic transport of mass, enthalpy, momentum, and solutes as well as stresses and deformation during solidification of aluminium. Alstruc deals with multicomponent alloys accounting for temperature dependent partition coefficients, liquidus slopes and the precipitation of secondary phases. The challenge associated with computation of microsegregation for multicomponent alloys is solved in Alstruc by approximating the phase diagram data by simple, analytical expressions which allows for a CPU-time efficient coupling with the macroscopic transport model. In the present work, the coupled model has been applied in a study of macrosegregation including thermal and solutal convection, solidification shrinkage and surface exudation on an industrial DC-cast billet.

  18. A METHOD FOR COMPARING MULTICOMPONENT, MULTIMEDIA POLLUTION ABATEMENT PROCESSES

    EPA Science Inventory

    The paper describes a comparison of the cost-effectiveness of four multicomponent, multimedia pollution abatement options: coal cleaning, limestone, limestone plus adipic acid, and coal cleaning plus limestone plus adipic acid. The comparison makes cost-effectiveness evaluations ...

  19. MPH: A library for distributed multi-component environment

    SciTech Connect

    Ding, Chris H.Q.; He, Yun

    2001-06-01

    Many current large and complex HPC applications are based on semi-independent program components developed by different groups or for different purposes. On distributed memory parallel supercomputers, how to perform component-name registration and initialize communications between independent components are among the first critical steps in establishing a distributed multi-component environment. Here we describe MPH, a multi-component handshaking library that resolves these tasks in a convenient and consistent way. MPH uses MPI for high performance and supports many PVM functionality. It supports two major parallel integration mechanism: multi-component multi-executable (MCME) and multi-component single-executable (MCME). It is a simple, easy-to-use module for developing practical codes, or as basis for larger software tools/frameworks.

  20. Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: Cometabolic inactivation of ammonia monooxygenase and substrate specificity

    SciTech Connect

    Rasche, M.E.; Hyman, M.R.; Arp, D.J. )

    1991-10-01

    The soil nitrifying bacterium Nitrosomonas europaea is capable of degrading trichloroethylene (TCE) and other halogenated hydrocarbons. TCE cometabolism by N. europaea resulted in an irreversible loss of TCE biodegradative capacity, ammonia-oxidizing activity, and ammonia-dependent O{sub 2} uptake by the cells. Inactivation was not observed in the presence of allylthiourea, a specific inhibitor of enzyme ammonia monooxygenase, or under anaerobic conditions, indicating that the TCE-mediated inactivation required ammonia monooxygenase activity. When N. europaea cells were incubated with ({sup 14}C)TCE under conditions which allowed turnover of ammonia monooxygenase, a number of cellular proteins were covalently labeled with {sup 14}C. Treatment of cells with allylthiourea or acetylene prior to incubation with ({sup 14}C)TCE prevented incorporation of {sup 14}C into proteins. The ammonia-oxidizing activity of cells inactivated in the presence of TCE could be recovered through a process requiring de novo protein synthesis. In addition to TCE, a series of chlorinated methanes, ethanes, and other ethylenes were screened as substrates for ammonia monooxygenase and for their ability to inactivate the ammonia-oxidizing system of N. europaea. The chlorocarbons would be divided into three classes depending on their biodegradability and inactivating potential: (1) compounds which were not biodegradable by N. europaea and which had no toxic effect on the cells (2) compounds which were cooxidized by N. europaea and had little or no toxic effect on the cells; and (3) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. europaea.

  1. Induction of cytochrome P450 1A1 and monooxygenase activity in Tilapia by sediment extract

    SciTech Connect

    Ueng, Y.F.; Ueng, T.H.; Liu, T.Y.

    1995-01-01

    Cytochrome P450 (P450)-dependent monooxygenases of fishes are inducible by a variety of environmental pollutants including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Induction of fish monoxygenases may serve as a biological monitor for PAH- and PCB-types of environmental chemicals. Many studies have demonstrated environmental induction of fish monooxygenases using various experimental approaches. However, relatively few studies have been conducted using fish treated with contaminated river sediment extracts. Damsui River is the largest river in the north of Taiwan. The lower section of the river in the Taipei Metropolitan area is heavily polluted by industrial and municipal wastes. Tilapia (Oreochromis mossambicus) is one of the few species of fish that occur in the polluted river. Previous field studies showed that the levels of P450 1A1, benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase activities in tilapia collected at Fu-Ho Bridge, a polluted section of Damsui River, were higher than respective levels in fish collected from an unpolluted section. These results suggested that tilapia caught at the polluted site were exposed to substances similar in action to PAHs and PCBs, because these chemical pollutants are potent inducers of P450 1A1. PAHs and PCBs are persistent compounds that can accumulate in sediment. Tilapia are occasionally associated with the bottom and could ingest chemically contaminated sediment. In the present study, we determined the induction properties of monooxygenases using tilapia treated with extract of sediment collected from a polluted section of Damsui River. The present study demonstrates that Damsui River sediment extract has the ability to induce hepatic P450 1A1 and dependent monooxygenase activities in tilapia. 17 refs., 2 figs., 2 tabs.

  2. Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Niewerth, Heiko

    2014-01-01

    A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule. PMID:25239889

  3. "Hot Tub Rash" and "Swimmer's Ear" (Pseudomonas)

    MedlinePlus

    Facts About “Hot Tub Rash” and “Swimmer’s Ear” (Pseudomonas) What is Pseudomonas and how can it affect me? Pseudomonas (sue-doh- ... a major cause of infections commonly known as “hot tub rash” and “swimmer’s ear.” This germ is ...

  4. Modulated decay in the multi-component Universe

    SciTech Connect

    Enomoto, Seishi; Kohri, Kazunori; Matsuda, Tomohiro E-mail: kohri@post.kek.jp

    2013-08-01

    The early Universe after inflation may have oscillations, kinations (nonoscillatory evolution of a field), topological defects, relativistic and non-relativistic particles at the same time. The Universe whose energy density is a sum of those components can be called the multi-component Universe. The components, which may have distinguishable density scalings, may decay modulated. In this paper we study generation of the curvature perturbations caused by the modulated decay in the multi-component Universe.

  5. Multicomponent reactions: A simple and efficient route to heterocyclic phosphonates.

    PubMed

    Haji, Mohammad

    2016-01-01

    Multicomponent reactions (MCRs) are one of the most important processes for the preparation of highly functionalized organic compounds in modern synthetic chemistry. As shown in this review, they play an important role in organophosphorus chemistry where phosphorus reagents are used as substrates for the synthesis of a wide range of phosphorylated heterocycles. In this article, an overview about multicomponent reactions used for the synthesis of heterocyclic compounds bearing a phosphonate group on the ring is given. PMID:27559377

  6. Multicomponent reactions: A simple and efficient route to heterocyclic phosphonates

    PubMed Central

    2016-01-01

    Summary Multicomponent reactions (MCRs) are one of the most important processes for the preparation of highly functionalized organic compounds in modern synthetic chemistry. As shown in this review, they play an important role in organophosphorus chemistry where phosphorus reagents are used as substrates for the synthesis of a wide range of phosphorylated heterocycles. In this article, an overview about multicomponent reactions used for the synthesis of heterocyclic compounds bearing a phosphonate group on the ring is given. PMID:27559377

  7. A Weibull characterization for tensile fracture of multicomponent brittle fibers

    NASA Technical Reports Server (NTRS)

    Barrows, R. G.

    1977-01-01

    A statistical characterization for multicomponent brittle fibers in presented. The method, which is an extension of usual Weibull distribution procedures, statistically considers the components making up a fiber (e.g., substrate, sheath, and surface) as separate entities and taken together as in a fiber. Tensile data for silicon carbide fiber and for an experimental carbon-boron alloy fiber are evaluated in terms of the proposed multicomponent Weibull characterization.

  8. Crystallization and initial crystallographic characterization of the Corynebacterium glutamicum nitrilotriacetate monooxygenase component A

    SciTech Connect

    Kim, Kyung-Jin; Kim, Sujin; Lee, Sujin; Kang, Beom Sik; Lee, Heung-Soo; Oh, Tae-Kwang; Kim, Myung Hee

    2006-11-01

    The Corynebacterium glutamicum NTA monooxygenase component A protein, which plays the central role in NTA biodegradation, was crystallized. The initial X-ray crystallographic characterization is reported. Safety and environmental concerns have recently dictated the proper disposal of nitrilotriacetate (NTA). Biodegradation of NTA is initiated by NTA monooxygenase, which is composed of two proteins: component A and component B. The NTA monooxygenase component A protein from Corynebacterium glutamicum was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate as the precipitant. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 111.04, b = 98.51, c = 171.61 Å, β = 101.94°. The asymmetric unit consists of four molecules, corresponding to a packing density of 2.3 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.

  9. Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.

    PubMed

    Watschinger, Katrin; Fuchs, Julian E; Yarov-Yarovoy, Vladimir; Keller, Markus A; Golderer, Georg; Hermetter, Albin; Werner-Felmayer, Gabriele; Hulo, Nicolas; Werner, Ernst R

    2012-04-01

    Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase. PMID:22220568

  10. Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase.

    PubMed

    Francis, Kevin; Gadda, Giovanni

    2009-10-01

    A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK(is)versus the ionic volume of the anion with slopes of 0.041+/-0.001 mM/A(3) and 0.027+/-0.001 mM/A(3) for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K(d) values of approximately 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme. PMID:19683782

  11. Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves).

    PubMed Central

    Bennett, R. N.; Hick, A. J.; Dawson, G. W.; Wallsgrove, R. M.

    1995-01-01

    The initial steps in glucosinolate biosynthesis are thought to proceed from amino acids, via N-hydroxy amino acids, to aldoximes. We showed previously that microsomes from green leaves of oilseed rape (Brassica napus cv Bienvenu) contain two distinct monooxygenases that catalyze the conversion of homophenylalanine and dihomomethionine to their respective aldoximes. Further characterization of these enzymes has now demonstrated that the latter enzyme catalyzes the NADPH-dependent oxidative decarboxylation of two higher homologs of methionine, in addition to dihomomethionine. No activity was found for either enzyme with L-methionine, DL-homomethionine, L-phenylalanine, L-tyrosine, or L-tryptophan. Both of these rape monooxygenase activities are dependent on O2, not requiring any other O2 species or radical. The presence of an unoxidized sulfur atom and its relative position in the side chain of the aliphatic substrates are important for binding to the active site of the methionine-homolog enzyme. Neither enzyme has any characteristics of a cytochrome P450-type enzyme, and antiserum raised against cytochrome P450 reductase did not significantly inhibit monooxygenase activity. PMID:12228596

  12. Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

    PubMed Central

    Krueger, Sharon K.; Williams, David E.

    2005-01-01

    Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a “soft-nucleophile”, usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics. In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences. The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration. PMID:15922018

  13. Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

    PubMed Central

    Watschinger, Katrin; Fuchs, Julian E.; Yarov-Yarovoy, Vladimir; Keller, Markus A.; Golderer, Georg; Hermetter, Albin; Werner-Felmayer, Gabriele; Hulo, Nicolas; Werner, Ernst R.

    2012-01-01

    Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure–function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis–Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase. PMID:22220568

  14. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    SciTech Connect

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K.

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  15. Inducible Monooxygenase Activities and 3-Methylcholanthrene-Initiated Tumorigenesis in Mouse Recombinant Inbred Sublines

    PubMed Central

    Atlas, Steven A.; Taylor, Benjamin A.; Diwan, Bhalchandra A.; Nebert, Daniel W.

    1976-01-01

    The induction of a certain group of hepatic monooxygenase activities by polycyclic aromatic compounds is regulated by the same locus or gene cluster controlling the formation of cytochrome P1–450 (P–448) in mice. Certain inbred strains of mice are "responsive" (Ahb) to such induction, whereas others are "nonresponsive" (Ahd). A pair of closely related sublines that differ with respect to the Ah locus (for aromatic hydrocarbon responsiveness) were used to identify or confirm the pleiotropic effects of this gene. The lines were derived by sibling-mating without selection from (C57L/J x AKR/J)F 2 mice; the two sublines were separated at the F12 generation. Ten microsomal monooxygenase activities and one cytosol enzyme activity known to be associated with the Ah locus were similarly associated with cytochrome P1–450 formation in these recombinant inbred sublines as well. Nine additional hepatic monooxygenase activities studied were found not to be associated with the Ah locus; certain of these activities were increased slightly, following treatment of nonresponsive as well as responsive mice with polycyclic aromatic compounds. The Ahb-containing subline was highly susceptible to 3-methylcholanthrene-induced subcutaneous sarcomas, whereas the Ah-d-containing subline was relatively resistant. These results emphasize the potential importance of this particular enzyme for the study of coordinated regulation in mammals. PMID:955403

  16. Quantitative surface spectroscopic analysis of multicomponent polymers

    NASA Astrophysics Data System (ADS)

    Zhuang, Hengzhong

    Angle-dependent electron spectroscopy for chemical analysis (ESCA) has been successfully used to examine the surface compositional gradient of a multicomponent polymer. However, photoelectron intensities detected at each take-off angle of ESCA measurements are convoluted signals. The convoluted nature of the signal distorts depth profiles for samples having compositional gradients. To recover the true concentration profiles for the samples, a deconvolution program has been described in Chapter 2. The compositional profiles of two classes of important multicomponent polymers, i.e., poly(dimethysiloxane urethane) (PU-DMS) segmented copolymers and fluorinated poly(amide urethane) block copolymers, are achieved using this program. The effects of the polymer molecular structure and the processing variation on its surface compositional profile have been studied. Besides surface composition, it is desirable to know whether the distribution of segment or block lengths at the surface is different than in the bulk, because this aspect of surface structure may lead to properties different than that predicted simply by knowledge of the surface composition and the bulk structure. In Chapter 3, we pioneered the direct determination of the distribution of polydimethylsiloxane (PDMS) segment lengths at the surface of PU-DMS using time-of-flight secondary ion mass spectrometry (SUMS). Exciting preliminary results are provided: for the thick film of PU-DMS with nominal MW of PDMS = 1000, the distribution of the PDMS segment lengths at the surface is nearly identical to that in the bulk, whereas in the case of the thick films of PU-DMS with nominal MW of PDMS = 2400, only those PDMS segments with MW of ca. 1000 preferentially segregated at the surface. As a potential minimal fouling coating or biocompatible cardio-vascular materials, PU-DMS copolymers eventually come into contact with water once in use. Could such an environmental change (from air to aqueous) induce any undesirable

  17. Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study.

    PubMed

    Putignani, Lorenza; Ambrosi, Cecilia; Ascenzi, Paolo; Visca, Paolo

    2004-01-01

    Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth. PMID:14684153

  18. Ti Multicomponent Alloy Bulks by Powder Metallurgy

    NASA Astrophysics Data System (ADS)

    Zhang, Kuibao; Wen, Guanjun; Dai, Hongchuan; Teng, Yuancheng; Li, Yuxiang

    2014-10-01

    In this study, CrCuFeMnMo0.5Ti multicomponent alloy bulks were prepared by powder metallurgy of mechanical alloying and sintering. A simple body-centered cubic (bcc) solid solution was prepared after 40 h ball milling of the raw CrCuFeMnMo0.5Ti metallic powder. Particles of the alloyed powder are in microsized structures, which are actually a soft agglomeration of lamellar grains with thicknesses less than 1 μm. Meanwhile, the lamellar granules are consisted of nanosized grains under rigid cold welding. The 80-h ball-milled powder was consolidated by cold pressing and subsequent sintering at 800°C. The observed main phase in the consolidated sample after milling for 80 h is still a bcc solid solution. The solidified sample of 80-h ball-milled powder exhibits a Vickers hardness of 468 HV, which is much higher than 171 HV of the counterpart prepared from the raw metallic powder.

  19. Mineral Selection for Multicomponent Equilibrium Geothermometry

    DOE PAGESBeta

    Plamer, C. D.; Ohly, S. R.; Smith, R. W.; Neupane, G.; McLing, T.; Mattson, E.

    2015-04-01

    Multicomponent geothermometry requires knowledge of the mineral phases in the reservoir with which the geothermal fluids may be equilibrated. These minerals phases are most often alteration products rather than primary minerals. We have reviewed the literature on geothermal systems representing most major geologic environments typically associated with geothermal activity and identified potential alteration products in various environments. We have included this information in RTEst, a code we have developed to estimate reservoir conditions (temperature, CO2 fugacity) from the geochemistry of near-surface geothermal waters. The information has been included in RTEst through the addition of filters that decrease the potential numbermore » of minerals from all possibilities based on the basis species to those that are more relevant to the particular conditions in which the user is interested. The three groups of filters include host rock type (tholeiitic, calc-alkaline, silicic, siliciclastic, carbonate), water type (acidic, neutral), and the temperature range over which the alteration minerals were formed (low, medium, high). The user-chosen mineral assemblage is checked to make sure that it does not violate the Gibbs phase rule. The user can select one of three mineral saturation weighting schemes that decrease the chance the optimization from being skewed by reaction stoichiometry or analytical uncertainty.« less

  20. Mineral Selection for Multicomponent Equilibrium Geothermometry

    SciTech Connect

    Plamer, C. D.; Ohly, S. R.; Smith, R. W.; Neupane, G.; McLing, T.; Mattson, E.

    2015-04-01

    Multicomponent geothermometry requires knowledge of the mineral phases in the reservoir with which the geothermal fluids may be equilibrated. These minerals phases are most often alteration products rather than primary minerals. We have reviewed the literature on geothermal systems representing most major geologic environments typically associated with geothermal activity and identified potential alteration products in various environments. We have included this information in RTEst, a code we have developed to estimate reservoir conditions (temperature, CO2 fugacity) from the geochemistry of near-surface geothermal waters. The information has been included in RTEst through the addition of filters that decrease the potential number of minerals from all possibilities based on the basis species to those that are more relevant to the particular conditions in which the user is interested. The three groups of filters include host rock type (tholeiitic, calc-alkaline, silicic, siliciclastic, carbonate), water type (acidic, neutral), and the temperature range over which the alteration minerals were formed (low, medium, high). The user-chosen mineral assemblage is checked to make sure that it does not violate the Gibbs phase rule. The user can select one of three mineral saturation weighting schemes that decrease the chance the optimization from being skewed by reaction stoichiometry or analytical uncertainty.

  1. An evaporation model of multicomponent solution drops

    NASA Astrophysics Data System (ADS)

    Sartori, Silvana; Liñán, Amable; Lasheras, Juan C.

    2010-11-01

    Solutions of polymers are widely used in the pharmaceutical industry as tablets coatings. These allow controlling the rate at which the drug is delivered, taste or appearance. The coating is performed by spraying and drying the tablets at moderate temperatures. The wetting of the coating solution on the pill's surface depends on the droplet Webber and Re numbers, angle of impact and on the rheological properties of the droplet. We present a model for the evaporation of multicomponent solutions droplets in a hot air environment with temperatures substantially lower than the boiling temperature of the solvent. As the liquid vaporizes from the surface the fluid in the drop increases in concentration, until reaching its saturation point. After saturation, precipitation occurs uniformly within the drop. As the surface regresses, a compacting front formed by the precipitate at its maximum packing density advances into the drop, while the solute continues precipitating uniformly. This porous shell grows fast due to the double effect of surface regression and precipitation. The evaporation rate is determined by the rates at which heat is transported to the droplet surface and at which liquid vapor diffuses away from it. When the drop is fully compacted, the evaporation is drastically reduced.

  2. Multicomponent assessment and treatment of cigarette pica.

    PubMed Central

    Goh, H L; Iwata, B A; Kahng, S W

    1999-01-01

    We conducted a multicomponent assessment and treatment for 4 individuals who engaged in cigarette pica. During Phase 1, three stimulus preference assessments were conducted to identify (a) the reinforcing component of the cigarette, (b) potential alternative reinforcers that may be used during treatment, and (c) whether the alternative reinforcer would compete effectively with cigarettes. Results were successful in identifying the reinforcing component of the cigarette and suggested the feasibility of using alternative reinforcers during treatment to eliminate cigarette pica. During Phase 2, the effects of two treatment procedures were evaluated. Noncontingent reinforcement (NCR) with the alternative edible reinforcer reduced the pica of 2 of the participants, but effects were not maintained when the initial dense schedule of NCR was thinned. Subsequently, differential reinforcement of alternative behavior with the alternative edible reinforcer was effective in reducing pica for 3 participants. An evaluation of nine treatment procedures failed to identify an effective intervention for the remaining participant; consequently, preventive measures were designed to minimize occurrences of cigarette pica. PMID:10513026

  3. Principles of Transport in Multicomponent Plasmas

    NASA Astrophysics Data System (ADS)

    Kaganovich, Igor D.; Franklin, Raoul N.; Demidov, Vladimir I.

    The main principles of transport in multicomponent plasmas are described. Because the bulk plasma is charged positively to keep electrons together with positive ions, negative ions are confined by electrostatic fields inside the plasma and they flow from the plasma periphery toward the center. It is shown that the flow velocity of negative ions is a nonlinear function of the negative ion density. Increasing the negative ion density makes the electron density profile flatter and leads to a decrease of the electric field. Such a nonlinear dependence of the negative ion flow velocity on their density results in the formation of steep gradients of negative ion density, or negative ion fronts. Addition of negative ions makes the plasma afterglow a complex process as well. Typically, two stages of afterglow appear. In the first stage, the negative ions are trapped inside the plasma and only electrons and positive ions can reach the walls. However, at a later time, electrons quickly leave the plasma, and the second stage of afterglow begins, in which electrons are totally absent and an ion-ion plasma forms. During this stage, only the negative and positive ions contribute to the wall fluxes. The complex structure of the radio frequency sheath in strongly electronegative gases is also reviewed. Similar phenomena are observed in dusty plasmas. A possible relevance to ball lightning is discussed.

  4. Single Pass Multi-component Harvester

    SciTech Connect

    Reed Hoskinson; J. Richard Hess

    2004-08-01

    Abstract. In order to meet the U. S. government’s goal of supplementing the energy available from petroleum by increasing the production of energy from renewable resources, increased production of bioenergy has become one of the new goals of the United States government and our society. U.S. Executive Orders and new Federal Legislation have mandated changes in government procedures and caused reorganizations within the government to support these goals. The Biomass Research and Development Initiative is a multi-agency effort to coordinate and accelerate all U.S. Federal biobased products and bioenergy research and development. The Initiative is managed by the National Biomass Coordination Office, which is staffed by both the DOE and the USDA. One of the most readily available sources of biomass from which to produce bioenergy is an agricultural crop residue, of which straw from small grains is the most feasible residue with which to start. For the straw residue to be used its collection must be energy efficient and its removal must not impact the sustainability of the growing environment. In addition, its collection must be economically advantageous to the producer. To do all that, a single pass multi-component harvester system is most desirable. Results from our first prototype suggest that current combines probably do adequate threshing and that a separate chassis can be developed that does additional separation and that is economically feasible.

  5. MPH: A Library for Distributed Multi-Component Environment

    Energy Science and Technology Software Center (ESTSC)

    2001-05-01

    A growing trend in developing large and complex applications on today's Teraflops compyters is to integrate stand-alone and/or semi-independent program components into a comprehensive simulation package. We develop MPH, a multi-component handshaking library that allows component models recognize and talk to each other in a convenient and consisten way, thus to run multi-component ulti-executable applications effectively on distributed memory architectures. MPH provides the following capabilities: component name registration, resource allocation, inter-component communication, inquiry on themore » multi-component environment, standard in/out redirect. It supports the following four integration mechanisms: Multi-Component Single-Executable (MCSE); Single-Component Multi-Executable (SCME); Multi-Component Multi-Executable (MCME); Multi-instance Multi-Executable (MIME). MPH currently works on IBM SP, SGI Origin, Compaq AlphaSC, Cray T3E, and PC clusters. It is being adopted in NCAR's CCSM and Colorado State University's icosahedra grid coupled model. A joint communicator between any two components could be created. MPI communication between local processors and remote processors are invoked through component names and the local id. More functions are available to inquire the global-id, local-id, number of executales, etc.« less

  6. Amine-phenyl multi-component gradient stationary phases.

    PubMed

    Dewoolkar, Veeren C; Kannan, Balamurali; Ashraf, Kayesh M; Higgins, Daniel A; Collinson, Maryanne M

    2015-09-01

    Continuous multi-component gradients in amine and phenyl groups were fabricated using controlled rate infusion (CRI). Solutions prepared from either 3-aminopropyltriethoxysilane (APTEOS) or phenyltrimethoxysilane (PTMOS) were infused, in a sequential fashion, at a controlled rate into an empty graduated cylinder housing a vertically aligned thin layer chromatography (TLC) plate. The hydrolyzed precursors reacted with an abundance of silanol (SiOH) groups on the TLC plates, covalently attaching the functionalized silane to its surface. The extent of modification by phenyl and amine was determined by the kinetics of each reaction and the exposure time at each point along the TLC plate. The local concentrations of phenyl and amine were measured using diffuse reflectance spectroscopy and X-ray photoelectron spectroscopy, respectively. The profile of the multi-component gradients strongly depended on the order of infusion, the direction of the gradient and the presence of available surface silanol groups. A slightly higher amount of phenyl can be deposited on the TLC plate by first modifying its surface with amine groups as they serve as a catalyst, enhancing condensation. Separation of water- and fat-soluble vitamins and the control of retention factors were demonstrated on the multi-component gradient TLC plates. Uniformly modified and single-component TLC plates gave different separations compared to the multi-component gradient plates. The retention factors of the individual vitamins depended on the order of surface modification, the spotting end, and whether the multi-component gradients align or oppose each other. PMID:26255112

  7. Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant Pseudomonas putida GS1.

    PubMed

    Mi, Jia; Schewe, Hendrik; Buchhaupt, Markus; Holtmann, Dirk; Schrader, Jens

    2016-07-01

    In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads' superior resilience compared to E. coli. Whole-cell P. putida harboring P450cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield YP/S of 79 %. To the authors' knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids. PMID:27263007

  8. [New Virulent Bacteriophages Active against Multiresistant Pseudomonas aeruginosa Strains].

    PubMed

    Balarjishvili, N Sh; Kvachadze, L I; Kutateladze, M I; Meskhi, T Sh; Pataridze, T K; Berishvili, T A; Tevdoradze, E Sh

    2015-01-01

    The sensitivity of 512 newly isolated Pseudomonas aeruginosa clinical strains to six classes of anti-microbial preparations has been studied. Antibiotic-resistant strains were selected and genotyped. Three new virulent bacteriophages of the families Myoviridae and Podoviridae were isolated against these strains. The parameters of the intracellular phage development cycle were established, and the influence of inactivating factors (temperature, pH, and UV exposure) on phage viability was studied. The molecular weight of the phage genome was determined. Phage DNA restriction analysis and polyacrylamide gel electrophoresis in the presence of envelope protein SDS were carried out. The plating efficacy of phages on 28 genetically distant antibiotic-resistant P. aeruginosa strains was studied. It was established that 26 of them were lysed by phages with a high efficacy. The range of antibacterial action of the studied phages and their mixtures on 427 multi-drug-resistant clinical isolates was assessed. It is shown that including these phages in one multicomponent preparation enhanced their lytic activity. PMID:26859962

  9. Cyclic Peptidomimetics and Pseudopeptides from Multicomponent Reactions

    NASA Astrophysics Data System (ADS)

    Wessjohann, Ludger A.; Rhoden, Cristiano R. B.; Rivera, Daniel G.; Vercillo, Otilie Eichler

    Multicomponent reactions (MCRs) that provide in the final product amides are suitable to produce peptides and peptide-like moieties. The Passerini and Staudinger reactions provide one amide bond, and the Ugi-four-component reaction generates two amides from three or even four (or more) components, respectively. The Ugi-reaction thus is most important to produce peptides and peptoids while the Passerini reaction is useful to generate depsipeptoid moieties. In order to produce cyclic peptides and pseudopeptides, the linear peptidic MCR products have to be cyclized, usually with the help of bifunctional or activatable building blocks. Orthogonal but cyclizable secondary functionalities that need no protection in isonitrile MCRs commonly include alkenes (for ring closing metathesis), azide/alkyne (for Huisgen click reactions) or dienes and enoates (Diels-Alder) etc. If MCR-reactive groups are to be used also for the cyclisation, monoprotected bifunctional building blocks are used and deprotected after the MCR, e.g. for Ugi reactions as Ugi-Deprotection-Cyclisation (UDC). Alternatively one of the former building blocks or functional groups generated by the MCR can be activated. Most commonly these are activated amides (from so-called convertible isonitriles) which can be used e.g. for Ugi-Activation-Cyclisation (UAC) protocols, or most recently for a simultaneous use of both strategies Ugi-Deprotection/Activation-Cyclisation (UDAC). These methods mostly lead to small, medicinally relevant peptide turn mimics. In an opposing strategy, the MCR is rather used as ring-closing reaction, thereby introducing a (di-)peptide moiety. Most recently these processes have been combined to use MCRs for both, linear precursor synthesis and cyclisation. These multiple MCR approaches allow the most efficient and versatile one pot synthesis of macrocyclic pseudopeptides known to date.

  10. Engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalyst for large scale biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid

    PubMed Central

    Seo, Joo-Hyun; Kim, Hwan-Hee; Jeon, Eun-Yeong; Song, Young-Ha; Shin, Chul-Soo; Park, Jin-Byung

    2016-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are able to catalyze regiospecific Baeyer-Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are usually difficult to express in a functional form in microbial cells and are rather unstable under process conditions hindering their large-scale applications. Thereby, we investigated engineering of the BVMO from Pseudomonas putida KT2440 and the gene expression system to improve its activity and stability for large-scale biotransformation of ricinoleic acid (1) into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) (3), which can be hydrolyzed into 11-hydroxyundec-9-enoic acid (5) (i.e., a precursor of polyamide-11) and n-heptanoic acid (4). The polyionic tag-based fusion engineering of the BVMO and the use of a synthetic promoter for constitutive enzyme expression allowed the recombinant Escherichia coli expressing the BVMO and the secondary alcohol dehydrogenase of Micrococcus luteus to produce the ester (3) to 85 mM (26.6 g/L) within 5 h. The 5 L scale biotransformation process was then successfully scaled up to a 70 L bioreactor; 3 was produced to over 70 mM (21.9 g/L) in the culture medium 6 h after biotransformation. This study demonstrated that the BVMO-based whole-cell reactions can be applied for large-scale biotransformations. PMID:27311560

  11. Engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalyst for large scale biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid.

    PubMed

    Seo, Joo-Hyun; Kim, Hwan-Hee; Jeon, Eun-Yeong; Song, Young-Ha; Shin, Chul-Soo; Park, Jin-Byung

    2016-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are able to catalyze regiospecific Baeyer-Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are usually difficult to express in a functional form in microbial cells and are rather unstable under process conditions hindering their large-scale applications. Thereby, we investigated engineering of the BVMO from Pseudomonas putida KT2440 and the gene expression system to improve its activity and stability for large-scale biotransformation of ricinoleic acid (1) into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) (3), which can be hydrolyzed into 11-hydroxyundec-9-enoic acid (5) (i.e., a precursor of polyamide-11) and n-heptanoic acid (4). The polyionic tag-based fusion engineering of the BVMO and the use of a synthetic promoter for constitutive enzyme expression allowed the recombinant Escherichia coli expressing the BVMO and the secondary alcohol dehydrogenase of Micrococcus luteus to produce the ester (3) to 85 mM (26.6 g/L) within 5 h. The 5 L scale biotransformation process was then successfully scaled up to a 70 L bioreactor; 3 was produced to over 70 mM (21.9 g/L) in the culture medium 6 h after biotransformation. This study demonstrated that the BVMO-based whole-cell reactions can be applied for large-scale biotransformations. PMID:27311560

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

    EPA Science Inventory

    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...

  13. Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase

    PubMed Central

    Ferroni, Felix Martin; Tolmie, Carmien; Smit, Martha Sophia; Opperman, Diederik Johannes

    2016-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the “in” position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the “variable” loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs. PMID:27472055

  14. A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Green, J; Dalton, H

    1989-01-01

    1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol. PMID:2497729

  15. Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase.

    PubMed

    Ferroni, Felix Martin; Tolmie, Carmien; Smit, Martha Sophia; Opperman, Diederik Johannes

    2016-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the "in" position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the "variable" loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs. PMID:27472055

  16. Sensor arrays from multicomponent micropatterned nanoparticles and graphene.

    PubMed

    Nagelli, Enoch; Naik, Rajesh; Xue, Yuhua; Gao, Yunxiang; Zhang, Mei; Dai, Liming

    2013-11-01

    A novel approach for multicomponent patterning metal/metal oxide nanoparticles on graphene was developed, which involves region-specific plasma treatment, followed by region-selective substrate-enhanced electroless deposition of Au nanoparticles and solution alkalization of ferrous chloride tetrahydrate in the presence of ammonia into Fe3O4 nanoparticles. The resultant Fe3O4/Au multicomponent micropatterned-graphene films were found to be highly selective sensor arrays for detecting low levels of chemical vapor molecules at ppm levels. This novel concept could be applied to the development of various multicomponent patterned nanomaterials for many potential applications, ranging from nanoscale region-specific chemical-/bio-sensor arrays to multifunctional optoelectronic devices. PMID:24113252

  17. Surface-slip equations for multicomponent nonequilibrium air flow

    NASA Technical Reports Server (NTRS)

    Gupta, R. N.; Scott, C. D.; Moss, J. N.

    1985-01-01

    Equations are presented for the surface-slip (or jump) values of species concentration, pressure, velocity, and temperature in the low-Reynolds number, high-altitude flight regime of a space vehicle. The equations are obtained from closed form solutions of the mass, momentum, and energy flux equations using the Chapman-Enskog velocity distribution function. This function represents a solution of the Boltzmann equation in the Navier-Stokes approximation. The analysis, obtained for nonequilibrium multicomponent air flow, includes the finite-rate surface catalytic recombination and changes in the internal energy during reflection from the surface. Expressions for the various slip quantities were obtained in a form which can be employed in flowfield computations. A consistent set of equations is provided for multicomponent, binary, and single species mixtures. Expression is also provided for the finite-rate, species-concentration boundary condition for a multicomponent mixture in absence of slip.

  18. Opportunities for Multicomponent Hybrid Hydrogels in Biomedical Applications

    PubMed Central

    2015-01-01

    Hydrogels provide mechanical support and a hydrated environment that offer good cytocompatibility and controlled release of molecules, and myriad hydrogels thus have been studied for biomedical applications. In the past few decades, research in these areas has shifted increasingly to multicomponent hydrogels that better capture the multifunctional nature of native biological environments and that offer opportunities to selectively tailor materials properties. This review summarizes recent approaches aimed at producing multicomponent hydrogels, with descriptions of contemporary chemical and physical approaches for forming networks, and of the use of both synthetic and biologically derived molecules to impart desired properties. Specific multicomponent materials with enhanced mechanical properties are presented, as well as materials in which multiple biological functions are imparted for applications in tissue engineering, cancer treatment, and gene therapies. The progress in the field suggests significant promise for these approaches in the development of biomedically relevant materials. PMID:25426888

  19. Surface-slip equations for multicomponent, nonequilibrium air flow

    NASA Technical Reports Server (NTRS)

    Gupta, Roop N.; Scott, Carl D.; Moss, James N.; Goglia, Gene

    1985-01-01

    Equations are presented for the surface slip (or jump) values of species concentration, pressure, velocity, and temperature in the low-Reynolds-number, high-altitude flight regime of a space vehicle. These are obtained from closed-form solutions of the mass, momentum, and energy flux equations using the Chapman-Enskog velocity distribution function. This function represents a solution of the Boltzmann equation in the Navier-Stokes approximation. The analysis, obtained for nonequilibrium multicomponent air flow, includes the finite-rate surface catalytic recombination and changes in the internal energy during reflection from the surface. Expressions for the various slip quantities have been obtained in a form which can readily be employed in flow-field computations. A consistent set of equations is provided for multicomponent, binary, and single species mixtures. Expression is also provided for the finite-rate species-concentration boundary condition for a multicomponent mixture in absence of slip.

  20. Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases.

    PubMed

    Beeson, William T; Phillips, Christopher M; Cate, Jamie H D; Marletta, Michael A

    2012-01-18

    Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O(2)-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose. PMID:22188218

  1. Overexpression and purification of the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Chan, Sunney I; Nguyen, H-Hoa T; Chen, Kelvin H-C; Yu, Steve S-F

    2011-01-01

    The particulate methane monooxygenase (pMMO) is a multi-copper enzyme that mediates the facile conversion of methane to methanol in methanotrophic bacteria. As a membrane-bound multi-subunit metalloprotein, the highly active protein has been difficult to isolate and purify to homogeneity for biochemical and biophysical studies. In this chapter, we describe a method to overexpress pMMO with good specific activity in high yields in the intracytoplasmic membranes of the host organism, together with two protocols to isolate and purify the enzyme from pMMO-enriched membranes without loss of the copper cofactors and enzymatic activity. PMID:21419922

  2. Metal reconstitution of particulate methane monooxygenase and heterologous expression of the pmoB subunit.

    PubMed

    Smith, Stephen M; Balasubramanian, Ramakrishnan; Rosenzweig, Amy C

    2011-01-01

    Particulate methane monooxygenase (pMMO) is a multisubunit metalloenzyme complex used by methanotrophic bacteria to oxidize methane in the first step of carbon assimilation and energy production. In this chapter, we detail methods to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. We also describe protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. These approaches were used to address fundamental questions concerning the metal content and location of the pMMO active site. PMID:21419923

  3. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

    PubMed

    Lo Leggio, Leila; Simmons, Thomas J; Poulsen, Jens-Christian N; Frandsen, Kristian E H; Hemsworth, Glyn R; Stringer, Mary A; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S; De Maria, Leonardo; Harris, Paul V; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes. PMID:25608804

  4. Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276.

    PubMed

    Saeki, H; Akira, M; Furuhashi, K; Averhoff, B; Gottschalk, G

    1999-07-01

    Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K(m) and Vmax values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 microM and 2.4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains. PMID:10439411

  5. Phylogenomics and systematics in Pseudomonas

    PubMed Central

    Gomila, Margarita; Peña, Arantxa; Mulet, Magdalena; Lalucat, Jorge; García-Valdés, Elena

    2015-01-01

    The genus Pseudomonas currently contains 144 species, making it the genus of Gram-negative bacteria that contains the largest number of species. Currently, multilocus sequence analysis (MLSA) is the preferred method for establishing the phylogeny between species and genera. Four partial gene sequences of housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) were obtained from 112 complete or draft genomes of strains related to the genus Pseudomonas that were available in databases. These genes were analyzed together with the corresponding sequences of 133 Pseudomonas type strains of validly published species to assess their correct phylogenetic assignations. We confirmed that 30% of the sequenced genomes of non-type strains were not correctly assigned at the species level in the accepted taxonomy of the genus and that 20% of the strains were not identified at the species level. Most of these strains had been isolated and classified several years ago, and their taxonomic status has not been updated by modern techniques. MLSA was also compared with indices based on the analysis of whole-genome sequences that have been proposed for species delineation, such as tetranucleotide usage patterns (TETRA), average nucleotide identity (ANIm, based on MUMmer and ANIb, based on BLAST) and genome-to-genome distance (GGDC). TETRA was useful for discriminating Pseudomonas from other genera, whereas ANIb and GGDC clearly separated strains of different species. ANIb showed the strongest correlation with MLSA. The correct species classification is a prerequisite for most diversity and evolutionary studies. This work highlights the necessity for complete genomic sequences of type strains to build a phylogenomic taxonomy and that all new genome sequences submitted to databases should be correctly assigned to species to avoid taxonomic inconsistencies. PMID:26074881

  6. Chromium reduction in Pseudomonas putida

    SciTech Connect

    Ishibashi, Y.; Cervantes, C.; Silver, S. )

    1990-07-01

    Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a K{sub m} of 40 {mu}M CrO{sub 4}{sup 2{minus}}. Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.

  7. Virasoro symmetry of the constrained multicomponent Kadomtsev-Petviashvili hierarchy and its integrable discretization

    NASA Astrophysics Data System (ADS)

    Li, Chuanzhong; He, Jingsong

    2016-06-01

    We construct Virasoro-type additional symmetries of a kind of constrained multicomponent Kadomtsev-Petviashvili (KP) hierarchy and obtain the Virasoro flow equation for the eigenfunctions and adjoint eigenfunctions. We show that the algebraic structure of the Virasoro symmetry is retained under discretization from the constrained multicomponent KP hierarchy to the discrete constrained multicomponent KP hierarchy.

  8. Thermal response of integral multicomponent composite thermal protection systems

    NASA Technical Reports Server (NTRS)

    Stewart, D. A.; Leiser, D. B.; Smith, M.; Kolodziej, P.

    1985-01-01

    Integral-multicomponent thermal-protection materials are discussed in terms of their thermal response to an arc-jet airstream. In-depth temperature measurements are compared with predictions from a one-dimensional, finite-difference code using calculated thermal conductivity values derived from an engineering model. The effect of composition, as well as the optical properties of the bonding material between components, on thermal response is discussed. The performance of these integral-multicomponent composite materials is compared with baseline Space Shuttle insulation.

  9. Rapid correction of electron microprobe data for multicomponent metallic systems

    NASA Technical Reports Server (NTRS)

    Gupta, K. P.; Sivakumar, R.

    1973-01-01

    This paper describes an empirical relation for the correction of electron microprobe data for multicomponent metallic systems. It evaluates the empirical correction parameter, a for each element in a binary alloy system using a modification of Colby's MAGIC III computer program and outlines a simple and quick way of correcting the probe data. This technique has been tested on a number of multicomponent metallic systems and the agreement with the results using theoretical expressions is found to be excellent. Limitations and suitability of this relation are discussed and a model calculation is also presented in the Appendix.

  10. Fast Rotating Scalar and Multi-component Bose Gases

    NASA Technical Reports Server (NTRS)

    Ho, TIn-Lun Jason

    2003-01-01

    We show that in the limit of large angular momentum, many equilibrium and dynamical phenomena of scalar and multi-component Bose gases can be accounted for by approximating the system to reside in an effective lowest Landau level. This method explains the origin of the mysterious stripe formation in fast rotating Bose gas recently observed at JILA, and accounts for all the dynamical details observed in this experiment. To further demonstrate the usefulness of this method, we present its predictions of the interference patterns of two vortex lattices, and rich vortex lattice structures in multi-component Bose gases.

  11. A Weibull characterization for tensile fracture of multicomponent brittle fibers

    NASA Technical Reports Server (NTRS)

    Barrows, R. G.

    1977-01-01

    Necessary to the development and understanding of brittle fiber reinforced composites is a means to statistically describe fiber strength and strain-to-failure behavior. A statistical characterization for multicomponent brittle fibers is presented. The method, which is an extension of usual Weibull distribution procedures, statistically considers the components making up a fiber (e.g., substrate, sheath, and surface) as separate entities and taken together as in a fiber. Tensile data for silicon carbide fiber and for an experimental carbon-boron alloy fiber are evaluated in terms of the proposed multicomponent Weibull characterization.

  12. Biodegradation of tetrahydrofuran and 1,4-dioxane by soluble diiron monooxygenase in Pseudonocardia sp. strain ENV478.

    PubMed

    Masuda, Hisako; McClay, Kevin; Steffan, Robert J; Zylstra, Gerben J

    2012-01-01

    1,4-Dioxane is an important groundwater contaminant. Pseudonocardia sp. strain ENV478 degrades 1,4-dioxane via cometabolism after the growth on tetrahydrofuran (THF) and other carbon sources. Here, we have identified a THF monooxygenase (thm) in ENV478. The thm genes are transcribed constitutively and are induced to higher levels by THF. Decreased translation of the thmB gene encoding one of the monooxygenase subunits by antisense RNA resulted in the loss of its ability to degrade THF and 1,4-dioxane. This is the first study to link thm genes to THF degradation, as well as the cometabolic oxidation of 1,4-dioxane. PMID:23147387

  13. Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.

    PubMed Central

    Neidle, E L; Hartnett, C; Ornston, L N; Bairoch, A; Rekik, M; Harayama, S

    1991-01-01

    The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified. PMID:1885518

  14. [Pseudomonas folliculitis after spa bath exposure].

    PubMed

    Uldall Pallesen, Kristine Appel; Andersen, Klaus Ejner; Mørtz, Charlotte Gotthard

    2012-06-25

    Pseudomonas aeruginosa is a rare cause of folliculitis. Pseudomonas folliculitis can develop after contact with contaminated water from swimming pools, hot tubs and spa baths. Systemic therapy may be indicated in patients with widespread lesions, systemic symptoms or in immunosuppressed patients. We describe a 23-year-old healthy woman who developed a pustular rash and general malaise after using a spa bath contaminated with Pseudomonas aeruginosa. Bacterial culture from a pustule confirmed Pseudomonas folliculitis and the patient was treated with ciprofloxacin with rapid good effect. PMID:22735119

  15. The Single Pass Multi-component Harvester

    SciTech Connect

    Reed Hoskinson; John R. Hess

    2004-08-01

    collection must be economically advantageous to the producer. To do all that, a single pass multi-component harvester system is most desirable. Results from our first prototype suggest that current combines probably do adequate threshing and that a separate chassis can be developed that does additional separation and that is economically feasible.

  16. Strategies for innovation in multicomponent reaction design.

    PubMed

    Ganem, Bruce

    2009-03-17

    By generating structural complexity in a single step from three or more reactants, multicomponent reactions (MCRs) make it possible to synthesize target compounds with greater efficiency and atom economy. The history of such reactions can be traced to the mid-19th century when Strecker first produced alpha-aminonitriles from the condensation of aldehydes with ammonia and hydrogen cyanide. Recently, academic chemists have renewed their interest in MCRs. In part, the pharmaceutical industry has fueled this resurgence because of the growing need to assemble libraries of structurally complex substances for evaluation as lead compounds in drug discovery and development programs. The application of MCRs to that increasingly important objective remains limited by the relatively small number of such reactions that can be broadly applied to prepare biologically relevant or natural-product-like molecular frameworks. We were interested in applying logic-based approaches, such as our single reactant replacement (SRR) approach, as a way both to improve known MCRs and to design new multiple-component routes to bioactive structures. This Account provides several examples that illustrate the use of SRR with known MCRs as starting points for synthetic innovation in this area. As part of our working hypothesis, we initially explored strategies for engineering improvements into known MCRs, either by increasing the dimensionality--that is, changing an n-component to an (n + 1)-component reaction--or broadening the scope of useful input structures, or both. By exhaustively applying retrosynthetic analysis to the cognate MCR to identify and exploit alternative entry points into the overall reaction manifold, we have devised several such re-engineered MCRs. Serendipitous findings have also augmented the yield of useful developments from our logic-inspired approach. In some cases, we have identified surprising links between different compound families that provide useful new entry points

  17. Increased monooxygenase activity associated with resistance to permethrin in Pediculus humanus capitis (Anoplura: Pediculidae) from Argentina.

    PubMed

    González Audino, P; Barrios, S; Vassena, C; Mougabure Cueto, G; Zerba, E; Picollo, M I

    2005-05-01

    We studied the profile of permethrin resistance in populations of head lice infesting children 6-12 yr old in schools and their homes in and around Buenos Aires, Argentina. Five permethrin-resistant populations with different levels of resistance were collected: Hogar Loyola (HL), Republica de Turquia (RT), Hogar Mitre (HM), Guardia de Honor (GH), and Ricardo Guiraldes (RG). One susceptible population, Bandera Argentina (BA), also was collected. Their level of resistance was evaluated, and results showed resistance ratios of 13 for HL, 16 for RT, 22 for HM, 61 for GH, and 69 for RG. To elucidate the possible involvement of the cytochrome P450 monooxygenase system in conferring permethrin resistance, ethoxycoumarin-O-deethylase (ECOD) activity was measured in abdomens of individual third instars and adults by using a fluorometric assay. The ECOD activity was lower in the susceptible BA population (4.7 ng per louse) than in the resistant ones (13.7 ng per louse for RG, 12.3 ng per louse for GH, 8.6 ng per louse for RT, and 8.2 ng per louse for HL). ECOD activity was significantly correlated with the level of resistance in the field populations (r = 0.97, P = 0.0009), suggesting a role for cytochrome monooxygenase P450 system in permethrin resistance by head louse, Pediculus humanus capitis De Geer. PMID:15962785

  18. P450monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium

    PubMed Central

    Syed, Khajamohiddin; Yadav, Jagjit S

    2012-01-01

    Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system (LDS), mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosporium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed. PMID:22624627

  19. Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase.

    PubMed

    Robinson, Reeder; Qureshi, Insaf A; Klancher, Catherine A; Rodriguez, Pedro J; Tanner, John J; Sobrado, Pablo

    2015-11-01

    The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites. PMID:26375201

  20. Steady-state kinetic analysis of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Green, J; Dalton, H

    1986-01-01

    A steady-state kinetic analysis of purified soluble methane mono-oxygenase of Methylococcus capsulatus (Bath) was performed. The enzyme was found to follow a concerted-substitution mechanism. Methane binds to the enzyme followed by NADH, which reacts to yield reduced enzyme and NAD+. The reduced enzyme-methane complex binds O2 to give a second ternary complex, which breaks down to release water and methanol. In this way the enzyme can control the supply of electrons to the active site to coincide with the arrival of methane. Product-inhibition studies (with propylene as substrate) supported the reaction mechanism proposed. Ki values for NAD+ and propylene oxide are reported. The Km for NADH varied from 25 microM to 300 microM, depending on the nature of the hydrocarbon substrate, and thus supports the proposed reaction sequence. With methane as substrate the Km values for methane, NADH and O2 were shown to be 3 microM, 55.8 microM and 16.8 microM respectively. With propylene as substrate the Km values for propylene, NADH and O2 were 0.94 microM, 25.2 microM and 12.7-15.9 microM respectively. Methane mono-oxygenase was shown to be well adapted to the oxidation of methane compared with other straight-chain alkanes. PMID:3098230

  1. Characterization and structural analysis of an active particulate methane monooxygenase trimer from Methylococcus capsulatus (Bath).

    PubMed

    Kitmitto, Ashraf; Myronova, Natalia; Basu, Piku; Dalton, Howard

    2005-08-23

    The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). We describe here the biochemical characterization of a stable and active purified pMMO hydroxylase (pMMO-H) and report a three-dimensional (3D) structure, determined by electron microscopy and single-particle analysis at 23 A resolution. Both biochemical and structural data indicate that pMMO hydroxylase is trimeric, with each monomer unit comprised of three polypeptides of 47, 26, and 23 kDa. Comparison of the recent crystal structure [Lieberman, R. L., and Rosenzweig, A. C. (2005) Nature 434, 177] of an uncharacterized pMMO-H complex with the three-dimensional (3D) structure determined here yielded a good match between the principal features and the organization of the enzyme monomers into trimers. The data presented here advance our current understanding of particulate methane monooxygenase function by the characterization of an active form of the enzyme and the corresponding 3D structure. PMID:16101279

  2. Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase.

    PubMed

    Jun, Se-Young; Lewis, Kevin M; Youn, Buhyun; Xun, Luying; Kang, ChulHee

    2016-06-01

    Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2 -dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA(2-) into EmoA identified an intricate hydrogen bond network connected to Tyr(71) , which should potentially lower its pKa. Tyr(71) , along with nearby Glu(70) and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channelling of FMNH2 . PMID:26928990

  3. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.

    PubMed

    Brummund, Jan; Müller, Monika; Schmitges, Thomas; Kaluzna, Iwona; Mink, Daniel; Hilterhaus, Lutz; Liese, Andreas

    2016-09-10

    Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα-ionone/molP450. PMID:27396939

  4. Investigation of the enzymology and pharmacology of novel substrates and inhibitors of dopamine beta-monooxygenase

    SciTech Connect

    Roberts, S.F.

    1987-01-01

    Dopamine beta-monooxygenase (DBM) was shown to catalyze the selenoxidation of 2-(phenylseleno)ethylamines, selenium-containing analogues of dopamine, by the normal monooxygenase pathway. The compounds 2-(phenylseleno)-ethylamine (PAESe), 2-(4'-hydroxyphenylseleno)ethylamine (pOH PAESe), and 1-(phenylseleno)-2-propylamine (Me PAESe) were synthesized and fully characterized as DBM substrates. Two other classes of compounds were investigated as potential alternate substrates for DBM. The possibility of stereoselective sulfonylation of 2-(phenylsulfenyl)- ethylamine (PAESO) was considered. A unique class of compounds, 2-(phenylthio)ethanols were designed and synthesized as DBM substrates but were found to be a novel class of potent competitive inhibitors of DBM with respect to tyramine. Preliminary experiments were also performed in an effort to demonstrate that the potent antihypertensive and indirect-acting sympathomimetic activity of 2-(phenylthio)ethylamine (PAES) was a result of DBM-oxygenation of this compound in vivo. The specific reserpine-sensitive uptake of (/sup 3/H)-norepinephrine into rat brain synaptosomes was demonstrated as was the synaptosomal conversion of (/sup 3/H)-dopamine to (/sup 3/H)-norepinephrine.

  5. Control of substrate access to the active site in methane monooxygenase.

    PubMed

    Lee, Seung Jae; McCormick, Michael S; Lippard, Stephen J; Cho, Uhn-Soo

    2013-02-21

    Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH-MMOB (hereafter termed H-B) complex. Here we remedy this deficiency by providing a crystal structure of H-B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α(2)β(2) interface of α(2)β(2)γ(2) MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle. PMID:23395959

  6. Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs.

    PubMed

    McIntyre, Neil R; Lowe, Edward W; Battistini, Matthew R; Leahy, James W; Merkler, David J

    2016-08-01

    Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals. PMID:26024288

  7. Effects of nutrition and alcohol on the microsomal monooxygenase system (MMS) of rat kidney

    SciTech Connect

    Ronis, M.; Huang, J.; Ingelman-Sundberg, M.; Badger, T.M. Arkansas Children's Hospital Research Center, Little Rock )

    1991-03-15

    Ethanol is a known inducer of the hepatic cytochrome P450 dependent microsomal monooxygenase system (MMS). As a consequence, ethanol intake affects the clearance and metabolism of many drugs and other xenobiotics including acetaminophen, enflurane, carbon tetrachloride and ethanol itself. The major ethanol inducible cytochrome P450 isozyme in the rat liver, CYP 2E1, has been well characterized. Much less is known concerning extrahepatic effects of ethanol on the monooxygenase system. In the current study, the effects of diet and alcohol were examined on MMS activities and cytochrome P450 expression in the kidneys of adult male Sprague-Dawley rats. Three diets containing no ethanol and two diets containing ethanol at 35% of total calories were studied. Renal MMS activities were measured using enzyme specific substrates and isozyme apoprotein levels were determined by Western blot analysis using antibodies directed against rat hepatic cytochrome P450s CYP 2E1, CYP 2A1 and CYP 3A2. Several diet and alcohol induced effects were observed, including a 5-fold diet-independent ethanol induction of CYP 2E1 cross reactive protein. No diet or ethanol effects were observed in levels of CYP 2A1 or CYP 3A2 cross reactive proteins.

  8. GROUNDWATER MASS TRANSPORT AND EQUILIBRIUM CHEMISTRY MODEL FOR MULTICOMPONENT SYSTEMS

    EPA Science Inventory

    A mass transport model, TRANQL, for a multicomponent solution system has been developed. The equilibrium interaction chemistry is posed independently of the mass transport equations which leads to a set of algebraic equations for the chemistry coupled to a set of differential equ...

  9. Optimal Multicomponent Analysis Using the Generalized Standard Addition Method.

    ERIC Educational Resources Information Center

    Raymond, Margaret; And Others

    1983-01-01

    Describes an experiment on the simultaneous determination of chromium and magnesium by spectophotometry modified to include the Generalized Standard Addition Method computer program, a multivariate calibration method that provides optimal multicomponent analysis in the presence of interference and matrix effects. Provides instructions for…

  10. Multicomponent Training of Teachers of Students with Severe Disabilities

    ERIC Educational Resources Information Center

    Brown, Phillip; Stephenson, Jennifer; Carter, Mark

    2014-01-01

    Over the last decade, the obligation of general and special educators to utilize evidence-based instructional practices has become more prominent. Research increasingly suggests the failure of didactic teacher training alone to ensure implementation with fidelity of these practices by teachers in their classrooms. Multicomponent training (MCT)…

  11. Criteria for Modeling in LES of Multicomponent Fuel Flow

    NASA Technical Reports Server (NTRS)

    Bellan, Josette; Selle, Laurent

    2009-01-01

    A report presents a study addressing the question of which large-eddy simulation (LES) equations are appropriate for modeling the flow of evaporating drops of a multicomponent liquid in a gas (e.g., a spray of kerosene or diesel fuel in air). The LES equations are obtained from the direct numerical simulation (DNS) equations in which the solution is computed at all flow length scales, by applying a spatial low-pass filter. Thus, in LES the small scales are removed and replaced by terms that cannot be computed from the LES solution and instead must be modeled to retain the effect of the small scales into the equations. The mathematical form of these models is a subject of contemporary research. For a single-component liquid, there is only one LES formulation, but this study revealed that for a multicomponent liquid, there are two non-equivalent LES formulations for the conservation equations describing the composition of the vapor. Criteria were proposed for selecting the multicomponent LES formulation that gives the best accuracy and increased computational efficiency. These criteria were applied in examination of filtered DNS databases to compute the terms in the LES equations. The DNS databases are from mixing layers of diesel and kerosene fuels. The comparisons resulted in the selection of one of the multicomponent LES formulations as the most promising with respect to all criteria.

  12. Electrostatic screening in nanostructures with multicomponent electron plasma

    NASA Astrophysics Data System (ADS)

    Kovalev, V. M.; Chaplik, A. V.

    2008-10-01

    Screening of the Coulumb interaction accounting for the Friedel oscillations in the structures with multicomponent low-dimensional electron plasma is considered. Calculations are made for nanotubes, double quantum wells (DQW) and superlattices. The binding energy of a donor in DQW is found as a function of the subbands occupation numbers.

  13. Essential Unidimensionality Examination for Multicomponent Scales: An Interrelationship Decomposition Approach

    ERIC Educational Resources Information Center

    Raykov, Tenko; Pohl, Steffi

    2013-01-01

    A procedure for examining essential unidimensionality in multicomponent measuring instruments is discussed. The method is based on an application of latent variable modeling and is concerned with the extent to which a common factor for all components of a given scale accounts for their correlations. The approach provides point and interval…

  14. SIMULATION OF CONTINUOUS-CONTACT SEPARATION PROCESSES: MULTICOMPONENT, ADIABATIC ABSORPTION

    EPA Science Inventory

    A new algorithm has been developed for the steady-state simulation of multicomponent, adiabatic absorption in packed columns. The system of differential model equations that describe the physical absorption process is reduced to algebraic equations by using a finite difference me...

  15. Genomics of Secondary Metabolism in Pseudomonas spp.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas is a heterogeneous genus of bacteria known for its ubiquity in natural habitats and its prolific production of secondary metabolites. The structurally diverse chemical structures produced by Pseudomonas spp. result from biosynthetic processes with unusual features that have revealed no...

  16. Pseudomonas blight discovered on raspberry in Watsonville

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the winter (February) of 2013, a field of raspberries in Watsonville was discovered to be infected with Pseudomonas syringae, the causal agent of Pseudomonas blight disease. This was the first documentation of this disease on raspberry in our region. The infection of raspberry plants is manifeste...

  17. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  18. Functional Identification of a Novel Gene, moaE, for 3-Succinoylpyridine Degradation in Pseudomonas putida S16

    PubMed Central

    Jiang, Yi; Tang, Hongzhi; Wu, Geng; Xu, Ping

    2015-01-01

    Microbial degradation of N-heterocyclic compounds, including xanthine, quinoline, nicotinate, and nicotine, frequently requires molybdenum hydroxylases. The intramolecular electron transfer chain of molybdenum hydroxylases consists of a molybdenum cofactor, two distinct [2Fe-2S] clusters, and flavin adenine dinucleotide. 3-Succinoylpyridine monooxygenase (Spm), responsible for the transformation from 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine, is a crucial enzyme in the pyrrolidine pathway of nicotine degradation in Pseudomonas. Our previous work revealed that the heterotrimeric enzyme (SpmA, SpmB, and SpmC) requires molybdopterin cytosine dinucleotide as a cofactor for their activities. In this study, we knocked out four genes, including PPS_1556, PPS_2936, PPS_4063, and PPS_4397, and found that a novel gene, PPS_4397 encoding moaE, is necessary for molybdopterin cytosine dinucleotide biosynthesis. Resting cell reactions of the moaE deletion mutant incubated with 3 g l−1 nicotine at 30 °C resulted in accumulation of 3-succinoylpyridine, and the strain complemented by the moaE gene regained the ability to convert 3-succinoylpyridine. In addition, reverse transcription-quantitative polymerase chain reaction analysis indicated that the transcriptional levels of the genes of moaE, spmA, and spmC of Pseudomonas putida S16 were distinctly higher when grown in nicotine medium than in glycerol medium. PMID:26304596

  19. Oxidation of nitrotoluenes by toluene dioxygenase: Evidence for a monooxygenase reaction

    SciTech Connect

    Robertson, J.B.; Spain, J.C. ); Haddock, J.D.; Gibson, D.T. )

    1992-08-01

    Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.

  20. Ferrofluid effect on Pseudomonas pyoverdine

    NASA Astrophysics Data System (ADS)

    Poiata, Antoniea; Vlahovici, Al.; Creanga, Dorina-Emilia

    2005-03-01

    The magnetic fluid effect on some pigmented pathogen germs has been investigated. The fluorescence of the pyoverdine pigment obtained from Pseudomonas aeruginosa strain, cultivated in the presence of different magnetic fluid concentrations, was enhanced by magnetic fluid concentrations of 0.0015-1 ml/l. The antimicrobial activity of pyoverdine, when tested by means of agar diffusimetric method against Sarcina lutea, was found increased for relatively high concentrations of magnetic fluid; in the case of Staphylococcus aureus the pyoverdine antimicrobial activity was not dependent on the magnetic fluid concentration.

  1. Pseudomonas--an opportunistic foe.

    PubMed

    Baillie, Jonathan

    2014-01-01

    An honest account of some of the lessons learned in how to protect patients, staff, and visitors, against waterborne Pseudomonas aeruginosa by effectively monitoring a large healthcare facility's water supply, identifying potential 'trigger points', harnessing the expertise of a multidisciplinary team, encouraging all staff to 'go the extra mile' preventatively, and above all, 'going beyond compliance', was provided by George McCracken, head of Estates Risk and Environment at the Belfast Health and Social Care Trust--in whose Royal Jubilee Maternity Hospital three young babies died after an outbreak of the bacteraemia in early 2012--at a recent Water Management Society conference. HEJ editor, Jonathan Baillie, reports. PMID:24516937

  2. Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation.

    PubMed

    Lee, Sang-Hyuk; Baek, Kiheon; Lee, Ju-Eun; Kim, Byung-Gee

    2016-04-01

    Tyrosinase is a binuclear copper-containing metalloprotein that leads the fast and regio-selective o-hydroxylation of monophenols to o-diphenols. However, the subsequent second oxidation to produce o-quinones, i.e., melanin precursors, from the o-diphenols has restricted its use to the production of functional o-diphenol derivatives. Herein, we present a combined strategy for the effective inhibition of melanin formation in tyrosinase reaction, which allows the use of tyrosinase as a monophenol monooxygenase. The o-diphenolic products were protected from being oxidized in the tyrosinase reaction by borate ions and L-ascorbic acid (LAA). Borate-o-diphenol complexes were favorable formed at high pH and consequentially protected the o-diphenolic products from the catecholase activity of tyrosinase. LAA not only directly reduced the byproduct, o-quinones, into o-diphenols but also assisted the completion of the tyrosinase reaction cycle by removing a hydroxyl group attached to the copper metal cluster at the active site of the met-form tyrosinase. The regio-selective o-hydroxylation of 7,4'-dihydroxyisoflavone (daidzein) to produce 7,3',4'-trihydroxyisoflavone (3'-ODI) was successfully carried out by whole E. coli cell biotransformation with heterologously expressed tyrosinase from Bacillus megaterium. The yield of this o-hydroxylation of 5 mM daidzein in one-pot 400 mL reaction was ca. 100% in 90 min and the productivity was 16.3 mg 3'-ODI · L(-1)  ·  h(-1)  ·  DCW mg(-1) , which is considerably higher than that of other monooxygenases. The method effectively abolished melanin synthesis, so that the o-diphenolic product remained stable without enzyme inactivation. Other monophenolic phytochemicals such as resveratrol and genistein could be subjected to the same strategy. After 1 h, 1 mM of genistein and resveratrol were both converted to orobol and piceatannol, respectively, with ca. 95% conversion yield. These results support the strong

  3. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  4. Variability in subpopulation formation propagates into biocatalytic variability of engineered Pseudomonas putida strains

    PubMed Central

    Lindmeyer, Martin; Jahn, Michael; Vorpahl, Carsten; Müller, Susann; Schmid, Andreas; Bühler, Bruno

    2015-01-01

    Pivotal challenges in industrial biotechnology are the identification and overcoming of cell-to-cell heterogeneity in microbial processes. While the development of subpopulations of isogenic cells in bioprocesses is well described (intra-population variability), a possible variability between genetically identical cultures growing under macroscopically identical conditions (clonal variability) is not. A high such clonal variability has been found for the recombinant expression of the styrene monooxygenase genes styAB from Pseudomonas taiwanensis VLB120 in solvent-tolerant Pseudomonas putida DOT-T1E using the alk-regulatory system from P. putida GPo1. In this study, the oxygenase subunit StyA fused to eGFP was used as readout tool to characterize the population structure in P. putida DOT-T1E regarding recombinant protein content. Flow cytometric analyses revealed that in individual cultures, at least two subpopulations with highly differing recombinant StyA-eGFP protein contents appeared (intra-population variability). Interestingly, subpopulation sizes varied from culture-to-culture correlating with the specific styrene epoxidation activity of cells derived from respective cultures (clonal variability). In addition, flow cytometric cell sorting coupled to plasmid copy number (PCN) determination revealed that detected clonal variations cannot be correlated to the PCN, but depend on the combination of the regulatory system and the host strain employed. This is, to the best of our knowledge, the first work reporting that intra-population variability (with differing protein contents in the presented case study) causes clonal variability of genetically identical cultures. Respective impacts on bioprocess reliability and performance and strategies to overcome respective reliability issues are discussed. PMID:26483771

  5. Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

    PubMed Central

    Volmer, Jan; Neumann, Christoph; Schmid, Andreas

    2014-01-01

    The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120ΔC parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents. PMID:25128338

  6. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  7. Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase from Pseudomonas fluorescens IP01†

    PubMed Central

    Dong, Xuesong; Fushinobu, Shinya; Fukuda, Eriko; Terada, Tohru; Nakamura, Shugo; Shimizu, Kentaro; Nojiri, Hideaki; Omori, Toshio; Shoun, Hirofumi; Wakagi, Takayoshi

    2005-01-01

    The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases. PMID:15774891

  8. Recent insights into copper-containing lytic polysaccharide mono-oxygenases.

    PubMed

    Hemsworth, Glyn R; Davies, Gideon J; Walton, Paul H

    2013-10-01

    Recently the role of oxidative enzymes in the degradation of polysaccharides by saprophytic bacteria and fungi was uncovered, challenging the classical model of polysaccharide degradation of being solely via a hydrolytic pathway. 3D structural analyses of lytic polysaccharide mono-oxygenases of both bacterial AA10 (formerly CBM33) and fungal AA9 (formerly GH61) enzymes revealed structures with β-sandwich folds containing an active site with a metal coordinated by an N-terminal histidine. Following some initial confusion about the identity of the metal ion it has now been shown that these enzymes are copper-dependent oxygenases. Here we assess recent developments in the academic literature, focussing on the structures of the copper active sites. We provide critical comparisons with known small-molecules studies of copper-oxygen complexes and with copper methane monoxygenase, another of nature's powerful copper oxygenases. PMID:23769965

  9. Cell Non-Autonomous Activation of Flavin-containing Monooxygenase Promotes Longevity and Healthspan

    PubMed Central

    Leiser, Scott F.; Fletcher, Marissa; Leonard, Alison; Primitivo, Melissa; Rintala, Nicholas; Ramos, Fresnida J.; Miller, Dana L.; Kaeberlein, Matt

    2016-01-01

    Stabilization of the hypoxia-inducible factor-1 (HIF-1) increases lifespan and healthspan in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in C. elegans through a cell non-autonomous signal to the intestine resulting in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This pro-longevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and necessary for DR-mediated lifespan extension, suggesting that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple lifespan-extending interventions in mice, suggesting that these enzymes may play a critical role in promoting health and longevity across phyla. PMID:26586189

  10. Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

    PubMed Central

    Garajova, Sona; Mathieu, Yann; Beccia, Maria Rosa; Bennati-Granier, Chloé; Biaso, Frédéric; Fanuel, Mathieu; Ropartz, David; Guigliarelli, Bruno; Record, Eric; Rogniaux, Hélène; Henrissat, Bernard; Berrin, Jean-Guy

    2016-01-01

    The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi. PMID:27312718

  11. Rv1894c Is a Novel Hypoxia-Induced Nitronate Monooxygenase Required for Mycobacterium tuberculosis Virulence

    PubMed Central

    Klinkenberg, Lee G.; Karakousis, Petros C.

    2013-01-01

    Tuberculosis is difficult to cure, requiring a minimum of 6 months of treatment with multiple antibiotics. Small numbers of organisms are able to tolerate the antibiotics and persist in the lungs of infected humans, but they still require some metabolic activity to survive. We studied the role of the hypoxia-induced Rv1894c gene in Mycobacterium tuberculosis virulence in guinea pigs, which develop hypoxic, necrotic granulomas histologically resembling those in humans and found this gene to be necessary for full bacillary growth and survival. We characterized the function of the encoded enzyme as a nitronate monooxygenase, which is needed to prevent the buildup of toxic products during hypoxic metabolism and is negatively regulated by the transcriptional repressor KstR. Future studies will focus on developing small-molecule inhibitors that target Rv1894c and its homologs, with the goal of killing persistent bacteria, thereby shortening the time needed to treat tuberculosis. PMID:23408846

  12. Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis.

    PubMed

    Gadda, Giovanni; Francis, Kevin

    2010-01-01

    Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented. PMID:19577534

  13. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

    PubMed Central

    Lo Leggio, Leila; Simmons, Thomas J.; Poulsen, Jens-Christian N.; Frandsen, Kristian E. H.; Hemsworth, Glyn R.; Stringer, Mary A.; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S.; De Maria, Leonardo; Harris, Paul V.; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J.; Walton, Paul H.

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes. PMID:25608804

  14. Searching for monooxygenases and hydrolases in bacteria from an extreme environment.

    PubMed

    da Cruz, Georgiana F; Angolini, Célio F F; de Oliveira, Luciana G; Lopes, Patrícia F; de Vasconcellos, Suzan P; Crespim, Elaine; de Oliveira, Valéria M; dos Santos Neto, Eugênio V; Marsaioli, Anita J

    2010-06-01

    Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities. PMID:20204614

  15. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.

    PubMed

    Li, Xin; Beeson, William T; Phillips, Christopher M; Marletta, Michael A; Cate, Jamie H D

    2012-06-01

    The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear. Here, we present structures of Neurospora crassa PMO-2 and PMO-3 at 1.10 and 1.37 Å resolution, respectively. In the structures, dioxygen species are found in the active sites, consistent with the proposed cleavage mechanism. Structural and sequence comparisons between PMOs also reveal that the enzyme substrate-binding surfaces contain highly varied aromatic amino acid and glycosylation positions. The structures reported here provide evidence for a wide range of PMO substrate recognition patterns in the plant cell wall, including binding modes that traverse multiple glucan chains. PMID:22578542

  16. Expression and characterization of a lytic polysaccharide monooxygenase from Bacillus thuringiensis.

    PubMed

    Zhang, Huiyan; Zhao, Yong; Cao, Hailong; Mou, Guangqing; Yin, Heng

    2015-08-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered oxidative enzymes that are capable of oxidative cleavage of recalcitrant polysaccharides such as chitin or cellulose. Despite the importance of LPMOs in biomass conversion and the large number of lpmo genes in microorganisms, only a few LPMOs have been well studied, and further characterization of these proteins is thus of interest. In this study, a chitin-active AA10 family LPMO from Bacillus thuringiensis subsp. kurstaki, BtLPMO10A, is described. This enzyme generates even-numbered oxidized oligosaccharides as the dominated products from crystalline chitin, however, interestingly, when colloidal chitin is used as the substrate, a ladder of oxidized oligosaccharides is observed. These results provide new insights into the action mode of LPMOs that may be affected by the substrates. PMID:25936286

  17. Discovery and characterization of a new family of lytic polysaccharide monooxygenases.

    PubMed

    Hemsworth, Glyn R; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2014-02-01

    Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs. PMID:24362702

  18. Direct electrochemistry of the hydroxylase of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Kazlauskaite, J; Hill, H A; Wilkins, P C; Dalton, H

    1996-10-15

    The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulatus (Bath) at a modified gold electrode giving rise to two waves at 4(+/- 10) mV and -386(+/- 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -25(+/- 14) mV and -433(+/- 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials. PMID:8917455

  19. Fourier transform infrared characterization of the azido complex of methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

    PubMed

    Lu, Shen; Sazinsky, Matthew H; Whittaker, James W; Lippard, Stephen J; Moënne-Loccoz, Pierre

    2005-03-30

    The azido complex formed in oxidized methane monooxygenase from Methylococcus capsulatus (Bath) was investigated with resonance Raman and FTIR techniques. These experiments show the presence of a nuas(NNN) at approximately 2077 cm-1 which splits to two components at 2059 and 2073 cm-1 with 15N14N2. The vibrational data are assigned to an azido complex bound terminally to one iron(III) at the diiron center. When the azido complex is illuminated at 15 K, a new nuas(NNN) is observed at 2136 cm-1 which is assigned to a photodissociated HN3 within the substrate pocket. We propose a model where an aqua ligand engages a hydrogen bond interaction with the 1N atom of the azido group and acts as at a proton donor during the photolysis process. PMID:15783178

  20. Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

    SciTech Connect

    Dumitru, Razvan; Jiang, Wen Zhi; Weeks, Donald P.; Wilson, Mark A.

    2009-08-28

    Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 {angstrom} resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.

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

    NASA Astrophysics Data System (ADS)

    Roccatano, Danilo

    2015-07-01

    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.

  2. Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis.

    PubMed

    Miao, Ji; Ling, Alisha V; Manthena, Praveen V; Gearing, Mary E; Graham, Mark J; Crooke, Rosanne M; Croce, Kevin J; Esquejo, Ryan M; Clish, Clary B; Vicent, David; Biddinger, Sudha B

    2015-01-01

    Despite the well-documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. Here, using non-biased profiling methods, we identify the enzyme flavin-containing monooxygenase 3 (Fmo3) to be a target of insulin. FMO3 produces trimethylamine N-oxide (TMAO), which has recently been suggested to promote atherosclerosis in mice and humans. We show that FMO3 is suppressed by insulin in vitro, increased in obese/insulin resistant male mice and increased in obese/insulin-resistant humans. Knockdown of FMO3 in insulin-resistant mice suppresses FoxO1, a central node for metabolic control, and entirely prevents the development of hyperglycaemia, hyperlipidemia and atherosclerosis. Taken together, these data indicate that FMO3 is required for FoxO1 expression and the development of metabolic dysfunction. PMID:25849138

  3. Hepatic microsomal monooxygenase activity in black-crowned night herons (BCNHS) from the Chesapeake basin

    USGS Publications Warehouse

    Melancon, M.J.; Rattner, B.A.; Rice, C.P.; Hines, R.K.; Eisemann, J.

    1992-01-01

    In a continuation of our studies on the use of hepatic cytochromes P450 as a biomarker for contaminant exposure, BCNH eggs were collected from Baltimore Harbor (BH) (n = 20), Washington National Zoo (WNZ) (n = 13) and Chincoteague National Wildlife Refuge (CNWR) (reference location) (n = 20). Eggs were artificially incubated and sacrificed at pipping. Livers were snap frozen in liquid nitrogen and stored at -80?C until assay. Hepatic microsomes were prepared by differential centrifugation of homogenates and assayed for protein, benzyloxy-resorufin-O-dealkylase, (BROD) ethoxyresorufinO-dealkylase (EROD) and pentoxyresorufin-O-dealkylase (PROD). Monooxygenase assays were run in triplicate using a computer-coupled fluorometric microwell plate scanner. Values for EROD and BROD, but not PROD, from BH and WNZ were significantly greater (approximately double) than those from CNWR. Organochlorine pesticide residues were much higher in carcasses from BH and WNZ as compared to CNWR. Carcasses are presently being analyzed for PCB congeners.

  4. Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose.

    PubMed

    Garajova, Sona; Mathieu, Yann; Beccia, Maria Rosa; Bennati-Granier, Chloé; Biaso, Frédéric; Fanuel, Mathieu; Ropartz, David; Guigliarelli, Bruno; Record, Eric; Rogniaux, Hélène; Henrissat, Bernard; Berrin, Jean-Guy

    2016-01-01

    The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi. PMID:27312718

  5. Fungal lytic polysaccharide monooxygenases bind starch and β-cyclodextrin similarly to amylolytic hydrolases.

    PubMed

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav; Abou Hachem, Maher

    2016-08-01

    Starch-binding modules of family 20 (CBM20) are present in 60% of lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative breakdown of starch, which highlights functional importance in LPMO activity. The substrate-binding properties of starch-active LMPOs, however, are currently unexplored. Affinities and binding-thermodynamics of two recombinant fungal LPMOs toward starch and β-cyclodextrin were shown to be similar to fungal CBM20s. Amplex Red assays showed ascorbate and Cu-dependent activity, which was inhibited in the presence of β-cylodextrin and amylose. Phylogenetically, the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities. PMID:27397613

  6. A novel non-hydrolytic protein from Pseudomonas oryzihabitans enhances the enzymatic hydrolysis of cellulose.

    PubMed

    Qin, Yi-Min; Tao, Heng; Liu, You-Yan; Wang, Yan-Dong; Zhang, Jing-Ru; Tang, Ai-Xing

    2013-10-10

    Several kinds of protein such as the expansin, expansin-like proteins and LPMOs (lytic polysaccharide monooxygenases) are known to exert enhancement effects on cellulase activity. In this study, a novel cellulase synergistic protein named POEP1 was purified from the culture filtrate of Pseudomonas oryzihabitans CGMCC 6169, and was homogeneous on SDS-PAGE with a molecular weight of 60kDa. Mass spectrometry analysis indicated that it was an unknown protein without sequence similarity to the expansin and expansin-like proteins. Evaluation of the enzymatic hydrolysis of filter paper revealed that POEP1 had no cellulase activity but displayed high synergistic activity of 364% at a cellulase concentration of 0.1FPU/g of filter paper. When a mixture containing 0.6FPU cellulase and 700μg POEP1 per g of cellulose was evaluated, the maximal sugar yield was achieved, which was 2.2-fold greater than that with the cellulase alone. POEP1 was found to have functional similarity to the expansin and expansin-like proteins, which could decrease both the hydrogen-bond intensity and crystallinity, and cause the filter paper disruption. This study provided evidence for the existence of novel bacterial proteins in nature serving the same function as expansin and expansin-like proteins. PMID:23916949

  7. Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene.

    PubMed Central

    Shields, M S; Montgomery, S O; Cuskey, S M; Chapman, P J; Pritchard, P H

    1991-01-01

    Pseudomonas cepacia G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho-monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes which were blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase, and TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol, and phenol hydroxylation. PMID:1892384

  8. Physiological analysis of the expression of the styrene degradation gene cluster in Pseudomonas fluorescens ST.

    PubMed

    Santos, P M; Blatny, J M; Di Bartolo, I; Valla, S; Zennaro, E

    2000-04-01

    The effects of different carbon sources on expression of the styrene catabolism genes in Pseudomonas fluorescens ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the beta-galactosidase reporter system. Expression of the promoter of the stySR operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the stySR transcript in P. fluorescens ST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated PstyA, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit beta-galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and beta-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the beta-galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon. PMID:10742204

  9. Elucidation of the 4-Hydroxyacetophenone Catabolic Pathway in Pseudomonas fluorescens ACB▿

    PubMed Central

    Moonen, Mariëlle J. H.; Kamerbeek, Nanne M.; Westphal, Adrie H.; Boeren, Sjef A.; Janssen, Dick B.; Fraaije, Marco W.; van Berkel, Willem J. H.

    2008-01-01

    The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to β-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone. PMID:18502868

  10. Methane monooxygenase gene expression mediated by methanobactin in the presence of mineral copper sources

    PubMed Central

    Knapp, Charles W.; Fowle, David A.; Kulczycki, Ezra; Roberts, Jennifer A.; Graham, David W.

    2007-01-01

    Methane is a major greenhouse gas linked to global warming; however, patterns of in situ methane oxidation by methane-oxidizing bacteria (methanotrophs), nature's main biological mechanism for methane suppression, are often inconsistent with laboratory predictions. For example, one would expect a strong relationship between methanotroph ecology and Cu level because methanotrophs require Cu to sustain particulate methane monooxygenase (pMMO), the most efficient enzyme for methane oxidation. However, no correlation has been observed in nature, which is surprising because methane monooxygenase (MMO) gene expression has been unequivocally linked to Cu availability. Here we provide a fundamental explanation for this lack of correlation. We propose that MMO expression in nature is largely controlled by solid-phase Cu geochemistry and the relative ability of Cu acquisition systems in methanotrophs, such as methanobactins (mb), to obtain Cu from mineral sources. To test this hypothesis, RT-PCR expression assays were developed for Methylosinus trichosporium OB3b (which produces mb) to quantify pMMO, soluble MMO (the alternate MMO expressed when Cu is “unavailable”), and 16S-rRNA gene expression under progressively more stringent Cu supply conditions. When Cu was provided as CuCl2, pMMO transcript levels increased significantly consistent with laboratory work. However, when Cu was provided as Cu-doped iron oxide, pMMO transcript levels increased only when mb was also present. Finally, when Cu was provided as Cu-doped borosilicate glass, pMMO transcription patterns varied depending on the ambient mb:Cu supply ratio. Cu geochemistry clearly influences MMO expression in terrestrial systems, and, as such, local Cu mineralogy might provide an explanation for methane oxidation patterns in the natural environment. PMID:17615240

  11. Transformation yields of chlorinated ethenes by a methanotrophic mixed culture expressing particulate methane monooxygenase.

    PubMed Central

    Anderson, J E; McCarty, P L

    1997-01-01

    Transformation yields for the aerobic cometabolic degradation of five chlorinated ethenes were determined by using a methanotrophic mixed culture expressing particulate methane monooxygenase (pMMO). Transformation yields (expressed as moles of chlorinated ethene degraded per mole of methane consumed) were 0.57, 0.25, 0.058, 0.0019, and 0.00022 for trans-1,2-dichloroethylene (t-DCE), vinyl chloride (VC), cis-1,2-dichloroethylene (c-DCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE), respectively. Degradation of t-DCE and VC was observed only in the presence of formate or methane, sources of reducing energy necessary for cometabolism. The t-DCE and VC transformation yields represented 35 and 15%, respectively, of the theoretical maximum yields, based on reducing-energy availability from methane dissimilation to carbon dioxide, exclusive of all other processes that require reducing energy. The yields for t-DCE and VC were 20 times greater than the yields reported by others for cells expressing soluble methane monooxygenase (sMMO). Transformation yields for c-DCE, TCE, and 1,1-DCE were similar to or less than those for cultures expressing sMMO. Although methanotrophic biotreatment systems have typically been designed to incorporate cultures expressing sMMO, these results suggest that pMMO expression may be highly advantageous for degradation of t-DCE or VC. It may also be much easier to maintain pMMO expression in treatment systems, because pMMO is expressed by all methanotrophs whereas sMMO is expressed only by type II methanotrophs under copper-limited conditions. PMID:9023946

  12. Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases.

    PubMed

    Bochevarov, Arteum D; Li, Jianing; Song, Woon Ju; Friesner, Richard A; Lippard, Stephen J

    2011-05-18

    The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites, yet the physical and chemical properties of their oxygenated intermediates, designated P*, H(peroxo), Q, and Q* in MMOH and ToMOH(peroxo) in a subclass of TMOH, ToMOH, are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss which changes could give rise to the different behavior of H(peroxo) and Q. In particular, analysis of multiple crystal structures reveals that T213 in MMOH and the analogous T201 in TMOH, located in the immediate vicinity of the active site, have different rotatory configurations. We study the rotational energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to which T213 and T201 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by the QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed Mössbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the ToMOD regulatory protein, which is essential for intermediate formation and protein functioning in the ToMO system, is advanced. We find that the low quadrupole splitting parameter in the Mössbauer spectrum observed for a ToMOH(peroxo) intermediate can be explained by protonation of the peroxo moiety, possibly stabilized by the T201 residue. Finally, similarities between the oxygen activation mechanisms of the monooxygenases and cytochrome P450 are discussed. PMID:21517016

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

    PubMed

    Sehlmeyer, Sven; Wang, Linzhu; Langel, Dorothee; Heckel, David G; Mohagheghi, Hoda; Petschenka, Georg; Ober, Dietrich

    2010-01-01

    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 and the

  14. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

    SciTech Connect

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.

    2010-01-08

    Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 {angstrom}) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 {angstrom}). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 {alpha}-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an {alpha}-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.

  15. Eukaryotic formylglycine-generating enzyme catalyses a monooxygenase type of reaction.

    PubMed

    Peng, Jianhe; Alam, Sarfaraz; Radhakrishnan, Karthikeyan; Mariappan, Malaiyalam; Rudolph, Markus Georg; May, Caroline; Dierks, Thomas; von Figura, Kurt; Schmidt, Bernhard

    2015-09-01

    C α-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 μM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor. PMID:26077311

  16. The biochemical mechanism of auxin biosynthesis by an arabidopsis YUCCA flavin-containing monooxygenase.

    PubMed

    Dai, Xinhua; Mashiguchi, Kiyoshi; Chen, Qingguo; Kasahara, Hiroyuki; Kamiya, Yuji; Ojha, Sunil; DuBois, Jennifer; Ballou, David; Zhao, Yunde

    2013-01-18

    Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from Escherichia coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-visible spectrum. We show that YUC6 uses NADPH and oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH(-) by NADPH. Subsequently, FADH(-) reacts with oxygen to form a flavin-C4a-(hydro)peroxy intermediate, which we show has a maximum absorbance at 381 nm in its UV-visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasi-stable C4a-(hydro)peroxyl FAD intermediates. The YUC6 intermediate has a half-life of ∼20 s whereas that of some FMOs is >30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants. PMID:23188833

  17. Form Follows Function: Structural and Catalytic Variation in the Class A Flavoprotein Monooxygenases

    PubMed Central

    Crozier-Reabe, Karen; Moran, Graham R.

    2012-01-01

    Flavoprotein monooxygenases (FPMOs) exhibit an array of mechanistic solutions to a common chemical objective; the monooxygenation of a target substrate. Each FPMO efficiently couples reduction of a flavin cofactor by NAD(P)H to oxygenation of the target substrate via a (hydro)peroxyflavin intermediate. This purpose of this review is to describe in detail the Class A flavoprotein hydroxylases (FPMO) in the context of the other FPMO classes (B–F). Both one and two component FPMOs are found in nature. Two-component enzymes require, in addition to the monooxygenase, the involvement of a reductase that first catalyzes the reduction of the flavin by NAD(P)H. The Class A and B FPMOs are single-component and manage to orchestrate the same net reaction within a single peptide. The Class A enzymes have, by some considerable margin, the most complete research record. These enzymes use choreographed movements of the flavin ring that facilitate access of the organic substrates to the active site, provide a means for interaction of NADPH with the flavin, offer a mechanism to sequester the dioxygen reduction chemistry from solvent and a means to release the product. The majority of the discrete catalytic events of the catalytic cycle can be observed directly in exquisite detail using spectrophotometric kinetic methods and many of the key mechanistic conclusions are further supported by structural data. This review attempts to compile each of the key observations made for both paradigm and newly discovered examples of Class A FPMOs into a complete catalytic description of one enzymatic turnover. PMID:23443084

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

    PubMed Central

    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

  19. Isolation of the monooxygenase complex from Rhipicephalus (Boophilus) microplus - clues to understanding acaricide resistance.

    PubMed

    Graham, Kirsty M; Sparagano, Olivier A E; Finn, Robert D

    2016-06-01

    The monooxygenase complex is composed of three key proteins, a cytochrome P450 (CYP), the cytochrome P450 oxidoreductase (CPR) and cytochrome b5 and plays a key role in the metabolism and detoxification of xenobiotic substances, including pesticides. In addition, overexpression of these components has been linked to pesticide resistance in several important vectors of disease. Despite this, the monooxygenase complex has not been isolated from the Southern cattle tick Rhipicephalus (Boophilus) microplus, a major disease vector in livestock. Using bioinformatics 115 transcriptomic sequences were analyzed to identify putative pesticide metabolizing CYPs. RACE-PCR was used to amplify the full length sequence of one CYP; CYP3006G8 which displays a high degree of homology to members of the CYP6 and 9 subfamilies, known to metabolize pyrethroids. mRNA expression levels of CYP3006G8 were investigated in 11 strains of R. microplus with differing resistance profiles by qPCR, the results of which indicated a correlation with pyrethroid metabolic resistance. In addition to this gene, the sequences for CPR and cytochrome b5 were also identified and subsequently isolated from R. microplus using PCR. CYP3006G8 is only the third CYP gene isolated from R. microplus and the first to putatively metabolize pesticides. The initial results of expression analysis suggest that CYP3006G8 metabolizes pyrethroids but further biochemical characterization is required to confirm this. Differences in the kinetic parameters of human and mosquito CPR in terms of NADPH binding have been demonstrated and could potentially be used to design species specific pesticides. Similar differences in the tick CPR would confirm that this is a characteristic of heamatophagous arthropods. PMID:26850353

  20. Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

    PubMed

    Hrycay, Eugene G; Bandiera, Stelvio M

    2015-01-01

    This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex. PMID:26002730

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

    PubMed Central

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

    2015-01-01

    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. PMID:26459905

  2. The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases*

    PubMed Central

    Crouch, Lucy I.; Labourel, Aurore; Walton, Paul H.; Davies, Gideon J.; Gilbert, Harry J.

    2016-01-01

    Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations. PMID:26801613

  3. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

    PubMed

    Yoshimoto, Naoko; Onuma, Misato; Mizuno, Shinya; Sugino, Yuka; Nakabayashi, Ryo; Imai, Shinsuke; Tsuneyoshi, Tadamitsu; Sumi, Shin-ichiro; Saito, Kazuki

    2015-09-01

    S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves. PMID:26345717

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

    PubMed

    Paspaliari, Dafni K; Loose, Jennifer S M; Larsen, Marianne H; Vaaje-Kolstad, Gustav

    2015-03-01

    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 multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results demonstrate that L. monocytogenes has a fully functional chitinolytic system. Both chitinases show substrate degradation rates similar to those of the nonprocessive endo-chitinase SmChiC from Serratia marcescens. Compared to the S. marcescens LPMO chitin-binding protein CBP21, LmLPMO10 shows a similar rate 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. The chitinases on the other hand are both abundantly secreted in the presence of chitin. Although LmLPMO10 is shown to promote chitin degradation in tandem with the chitinases in vitro, the secretome and transcription data question whether this is the primary role of LmLPMO10 in vivo. PMID:25565565

  5. Electrostatic twisted modes in multi-component dusty plasmas

    NASA Astrophysics Data System (ADS)

    Ayub, M. K.; Ali, S.; Ikram, M.

    2016-01-01

    Various electrostatic twisted modes are re-investigated with finite orbital angular momentum in an unmagnetized collisionless multi-component dusty plasma, consisting of positive/negative charged dust particles, ions, and electrons. For this purpose, hydrodynamical equations are employed to obtain paraxial equations in terms of density perturbations, while assuming the Gaussian and Laguerre-Gaussian (LG) beam solutions. Specifically, approximated solutions for potential problem are studied by using the paraxial approximation and expressed the electric field components in terms of LG functions. The energy fluxes associated with these modes are computed and corresponding expressions for orbital angular momenta are derived. Numerical analyses reveal that radial/angular mode numbers as well as dust number density and dust charging states strongly modify the LG potential profiles attributed to different electrostatic modes. Our results are important for understanding particle transport and energy transfer due to wave excitations in multi-component dusty plasmas.

  6. Efficient Ab initio Modeling of Random Multicomponent Alloys

    NASA Astrophysics Data System (ADS)

    Jiang, Chao; Uberuaga, Blas P.

    2016-03-01

    We present in this Letter a novel small set of ordered structures (SSOS) method that allows extremely efficient ab initio modeling of random multicomponent alloys. Using inverse II-III spinel oxides and equiatomic quinary bcc (so-called high entropy) alloys as examples, we demonstrate that a SSOS can achieve the same accuracy as a large supercell or a well-converged cluster expansion, but with significantly reduced computational cost. In particular, because of this efficiency, a large number of quinary alloy compositions can be quickly screened, leading to the identification of several new possible high-entropy alloy chemistries. The SSOS method developed here can be broadly useful for the rapid computational design of multicomponent materials, especially those with a large number of alloying elements, a challenging problem for other approaches.

  7. Dropwise ignition versus external ignition for multicomponent fuel sprays

    NASA Astrophysics Data System (ADS)

    Mawid, M.; Aggarwal, S. K.

    1988-07-01

    An attempt has been made to identify conditions for dropwise ignition and spray ignition. Both pure as well as multicomponent fuels are considered. For dropwise ignition, an existing ignition criterion has been modified to account for the nonlinear dependence of reaction rate on fuel and oxygen concentrations and to account for the multicomponent nature of the fuel. The external or spray ignition is considered through the zero heat flux condition at the ignition source. The effect of chemical kinetics is examined by employing reaction schemes with unity as well as non-unity exponents of fuel and oxygen concentrations. Results indicate that for most of the conditions considered, the individual droplet ignition is favored over the external ignition. Only when the drop diameter is smaller than 30 microns, the spray ignites earlier than droplets. The addition of a small amount of a volatile component significantly enhances the ignitability of both modes. However, the effect is stronger for the dropwise ignition mode.

  8. Multicomponent delirium prevention: not as effective as NICE suggest?

    PubMed

    Teale, Elizabeth; Young, John

    2015-11-01

    Multicomponent delirium prevention strategies have been shown in intervention studies consistently to reduce the occurrence of delirium. Based on this convincing evidence base, the National Institute for Health and Care Excellence has advocated the widespread adoption of multicomponent delirium prevention interventions into the routine inpatient care of older people. However, despite successful reductions in incident delirium of about a third, anticipated reductions in mortality or admissions to long-term care--both clinically important endpoints statistically correlated with the occurrence of delirium--have not been conclusively observed. We hypothesise that the reasons for this disconnection are partly methodological, due to difficulties in delirium detection and blinding of study personnel to the intervention, but predominantly due to the underlying relationship between delirium and the abnormal health state of frailty; the interaction between these two geriatric syndromes is currently poorly understood. PMID:26316509

  9. Method for producing nanocrystalline multicomponent and multiphase materials

    DOEpatents

    Eastman, Jeffrey A.; Rittner, Mindy N.; Youngdahl, Carl J.; Weertman, Julia R.

    1998-01-01

    A process for producing multi-component and multiphase nanophase materials is provided wherein a plurality of elements are vaporized in a controlled atmosphere, so as to facilitate thorough mixing, and then condensing and consolidating the elements. The invention also provides for a multicomponent and multiphase nanocrystalline material of specified elemental and phase composition having component grain sizes of between approximately 1 nm and 100 nm. This material is a single element in combination with a binary compound. In more specific embodiments, the single element in this material can be a transition metal element, a non-transition metal element, a semiconductor, or a semi-metal, and the binary compound in this material can be an intermetallic, an oxide, a nitride, a hydride, a chloride, or other compound.

  10. Multi-component transparent conducting oxides: progress in materials modelling

    NASA Astrophysics Data System (ADS)

    Walsh, Aron; Da Silva, Juarez L. F.; Wei, Su-Huai

    2011-08-01

    Transparent conducting oxides (TCOs) play an essential role in modern optoelectronic devices through their combination of electrical conductivity and optical transparency. We review recent progress in our understanding of multi-component TCOs formed from solid solutions of ZnO, In2O3, Ga2O3 and Al2O3, with a particular emphasis on the contributions of materials modelling, primarily based on density functional theory. In particular, we highlight three major results from our work: (i) the fundamental principles governing the crystal structures of multi-component oxide structures including (In2O3)(ZnO)n and (In2O3)m(Ga2O3)l(ZnO)n; (ii) the relationship between elemental composition and optical and electrical behaviour, including valence band alignments; (iii) the high performance of amorphous oxide semiconductors. On the basis of these advances, the challenge of the rational design of novel electroceramic materials is discussed.

  11. Thin multicomponent films for functional enzyme devices and bioreactor particles

    PubMed Central

    Rusling, James F.; Wasalathanthri, Dhanuka P.; Schenkman, John B.

    2014-01-01

    Complex functional films containing enzymes and other biomolecules are easily fabricated in nm-scale thicknesses by using layer-by-layer (LbL) methodologies first popularized by Lvov and Decher. In this review, we highlight the high level functional capabilities possible with LbL films of biomolecules based on our own research experiences. We first describe the basics of enzyme film fabrication by LbL alternate electrostatic adsorption, then discuss how to make functional enzyme-polyion films of remarkably high stability. Focusing on cytochrome P450s, we discuss films developed to electrochemically activate the natural catalytic cycle of these key metabolic enzymes. We then describe multifunctional, multicomponent DNA/enzyme/polyion films on arrays and particle surfaces for high throughput metabolic toxicity screening using electrochemiluminescence and LC-MS/MS. Using multicomponent LbL films, complex functionality for bioanalytical and biochemical purposes can be achieved that is difficult or impossible using conventional approaches. PMID:25209428

  12. Force measurements in aerodynamics using piezoelectric multicomponent force transducers

    NASA Astrophysics Data System (ADS)

    Schewe, G.

    The present paper is concerned with a device for the measurement of steady and unsteady aerodynamic forces in a wind tunnel test. The paper represents a continuation of an article written by Schewe (1982) about a multicomponent balance consisting of piezoelectric force transducers for a high-pressure wind tunnel. Advantages of the piezoelectric force-measuring technique compared to other techniques are related to the high rigidity of the quartz crystal sensor elements, taking into account low interference (cross talk) for multicomponent measurements, high natural frequency, and broad dynamic range. It is pointed out that the limitations with respect to quasi-static measurements imposed by the drift of the zero point are not as extensive as generally believed, while drift correction methods improve the measurement accuracy.

  13. Transport equations for multicomponent anisotropic space plasmas - A review

    NASA Technical Reports Server (NTRS)

    Barakat, A. R.; Schunk, R. W.

    1982-01-01

    An attempt is made to present a unified approach to the study of transport phenomena in multicomponent anisotropic space plasmas. In particular, a system of generalized transport equations is presented that can be applied to widely different plasma flow conditions. The generalized transport equations can describe subsonic and supersonic flows, collision-dominated and collisionless flows, plasma flows in rapidly changing magnetic field configurations, multicomponent plasma flows with large temperature differences between the interacting species, and plasma flows that contain anisotropic temperature distributions. In addition, if Maxwell's equations of electricity and magnetism are added to the system of transport equations, they can be used to model electrostatic shocks, double layers, and magnetic merging processes. These transport equations also contain terms which act to regulate both the heat flow and temperature anisotropy, processes which appear to be operating in the solar wind.

  14. Efficient Ab initio Modeling of Random Multicomponent Alloys.

    PubMed

    Jiang, Chao; Uberuaga, Blas P

    2016-03-11

    We present in this Letter a novel small set of ordered structures (SSOS) method that allows extremely efficient ab initio modeling of random multicomponent alloys. Using inverse II-III spinel oxides and equiatomic quinary bcc (so-called high entropy) alloys as examples, we demonstrate that a SSOS can achieve the same accuracy as a large supercell or a well-converged cluster expansion, but with significantly reduced computational cost. In particular, because of this efficiency, a large number of quinary alloy compositions can be quickly screened, leading to the identification of several new possible high-entropy alloy chemistries. The SSOS method developed here can be broadly useful for the rapid computational design of multicomponent materials, especially those with a large number of alloying elements, a challenging problem for other approaches. PMID:27015491

  15. Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and b-cryptoxanthin by ferret carotene-9, 10-monooxygenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xanthophyll carotenoids, such as lutein, zeaxanthin and b-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,150-monooxygenase (CMO1) h...

  16. Symbiotic solitons in heteronuclear multicomponent Bose-Einstein condensates

    SciTech Connect

    Perez-Garcia, Victor M.; Beitia, Juan Belmonte

    2005-09-15

    We show that bright solitons exist in quasi-one-dimensional heteronuclear multicomponent Bose-Einstein condensates with repulsive self-interaction and attractive interspecies interaction. They are remarkably robust to perturbations of initial data and collisions and can be generated by the mechanism of modulational instability. Some possibilities for control and the behavior of the system in fully three-dimensional scenarios are also discussed.

  17. Efficient Isocyanide-less Isocyanide-Based Multicomponent Reactions

    PubMed Central

    Neochoritis, Constantinos G.; Stotani, Silvia; Mishra, Bhupendra; Dömling, Alexander

    2016-01-01

    Isocyanides are the “Jekyll and Hyde” of organic chemistry allowing for extremely interesting transformations that are not only extremely odorous but also noxious. Therefore, an isocyanide-less isocyanide-based multicomponent reaction (IMCR) has been developed, and this protocol is expected to replace many of the old procedures in the future not only in IMCR but in other areas of organic chemistry as well. PMID:25824100

  18. Thermionic evaporation of films of multicomponent chalcogenide semiconductors

    SciTech Connect

    Serigenko, T.I.; Gritsenko, K.P.; Kryuchin, A.A.; Petrov, V.V.; Yudin, G.Y.

    1985-08-01

    This paper describes a procedure for the preparation of thin films of multicomponent chalcogenide semiconductors of As-Te-Se and As-Te-Ge by thermionic evaporation, using a discrete evaporator. Films of thickness 20-60 nm evaporated onto a glass substrate had an adhesion of 35-40 kg/cm/sup 2/. The films have enhanced homogeneity and time stability as compared to thin films of the same composition prepared by thermal evaporation.

  19. Structure and compositional studies of multi-component nanoparticles

    NASA Astrophysics Data System (ADS)

    Malyavanatham, Gokul

    The laser ablation of microparticle (LAM) process was used to study nanoparticles of multi-component materials. The production process utilized laser ablation of a continuously flowing aerosol of micron-sized particles under a gas ambient. An aerosol generator entrained microparticles into a gas flow and directed them through a nozzle into a laser interaction cell. After plasma breakdown, the shock wave propagated through the microparticles and the nanoparticles condensed behind this shockwave. Two methods were developed to collect nanoparticles; the first method used supersonic impaction on substrates at room temperature to enable direct writing of thick films and the second method used electric fields to deflect and collect charged, individual nanoparticles. Two methods for generating multi-component nanostructured materials were studied. The first method involved feeding single-phase microparticles containing the desired composition. Lead Zirconate Titanate (PZT) microparticles were used to generate nanoparticles that were then impacted onto substrates to produce thick films. Quality, morphology, crystallization and composition variations of these thick films were analyzed using material characterization techniques. Segregation of elements and an overall deficiency in Zr and Ti were observed in the deposited thick films as a result of the agglomerated state of the PZT microparticles. However, the analysis for this material system was complicated by the presence of multiple compounds. To develop a further understanding of how segregation occurs in multi-component systems during the LAM process, a second method for generating multi-component nanoparticles by feeding mixtures of single component microparticles was studied. Nanoparticles generated by ablation of Cu and Au microparticle mixtures were collected electrostatically and analyzed. Interactions between exploding microparticles resulted in condensation of nanoparticles that were non-equilibrium solid

  20. Numerical study of multicomponent droplet vaporization at near critical conditions

    NASA Technical Reports Server (NTRS)

    Hsieh, Kwang-Chung; Shuen, Jian-Shun; Yang, Vigor

    1988-01-01

    A comprehensive numerical analysis of multicomponent droplet vaporization at near critical conditions has been carried out. The model is based on the full time-dependent conservation equations and accommodates various important high-pressure phenomena. As an example, the case involving a two-component (n-pentane and n-octane) fuel droplet in nitrogen gas is studied. The influences of transient effects, surface regression, ambient gas solubility, and phase-equilibrium relations on vaporization mechanisms are examined in detail.

  1. Design and minimum reflux calculations for multicomponent reactive distillation columns

    SciTech Connect

    Barbosa, D.; Doherty, M.F.

    1987-01-01

    A new set of transformed composition variables is introduced to simplify the design equations for single-feed, multicomponent reactive distillation columns. Based on these equations, a general method of calculating minimum reflux ratios for reactive distillation columns is presented. The new composition variables are also used to derive simple relationships between the dependent design variables, which are not evident when the design equations are written in terms of mole fractions.

  2. Multi-component generalization of the Camassa-Holm equation

    NASA Astrophysics Data System (ADS)

    Xia, Baoqiang; Qiao, Zhijun

    2016-09-01

    In this paper, we propose a multi-component system of the Camassa-Holm equation, denoted by CH(N , H), with 2 N components and an arbitrary smooth function H. This system is shown to admit Lax pair and infinitely many conservation laws. We particularly study the case N = 2 and derive the bi-Hamiltonian structures and peaked soliton (peakon) solutions for some examples.

  3. Multi-component stress history measurements and analysis

    SciTech Connect

    Stout, R.B.; Larson, D.B.

    1987-08-01

    Piezoresistance foil gages were tested dynamically in multi-component stress-strain experiments in order that the actual shock wave conditions of underground nuclear testing could be more closely simulated. The multi-component stress-strain histories were created in polymethylmethacrylate (PMMA) by using chemical explosions to generate spherical shock waves. In addition to the resistivity measurements from the foil gages, particle velocity was also measured at several radial positions from the explosion to provide a complete set of data for analysis. The gage interpretation (inverse) problem for multi-component stress-strain fields requires obtaining a sufficient number of independent measurements so that the different stress-strain components influencing the gage response can be uniquely inferred. The piezoresistance measurements provided data from a triple material foil gage and from ytterbium foil gages (bare gages). An analysis shows that the triple material gage containing foils of ytterbium, manganin, and constantan provided three independent resistivity measurements for the gage oriented in a perpendicular direction relative to the radial propagating shock front. An analysis of the ytterbium foil gages, which were tested in both perpendicular (normal) and parallel (tangential) directions relative to the radial shock front, show the resistivity responses from these two orientations are independent measurements. The results from the analyses of the gages compared well with experimental data. This analysis shows clearly that the material properties of the foil, the dimensions of the foil, and the material surrounding the foil greatly influence the total resistivity response of foil gages in a multi-component stress-strain field. 25 refs., 16 figs.

  4. Gouy Interferometry: Properties of Multicomponent System Omega Graphs

    SciTech Connect

    Miller, D G

    2007-01-24

    We consider the properties of {Omega} graphs ({Omega} vs f(z)) obtained from Gouy interferometry on multicomponent systems with constant diffusion coefficients. We show that they must have (a) either a maximum or else a minimum between f(z)=0 and f(z)=1 and (b) an inflection point between the f(z) value at the extremum and f(z)=1. Consequently, an {Omega} graph cannot have both positive and negative {Omega} values.

  5. Dynamic simulation of multicomponent reaction transport in water distribution systems.

    PubMed

    Munavalli, G R; Mohan Kumar, M S M S

    2004-04-01

    Given the presence of nutrients, regrowth of bacteria within a distribution system is possible. The bacterial growth phenomena, which can be studied by developing a multicomponent (substrate, biomass and disinfectant) reaction transport model, is governed by its relationship with the substrate (organic carbon) and disinfectant (chlorine). The multicomponent reaction transport model developed in the present study utilizes the simplified expressions for the basic processes (in bulk flow and at pipe wall) such as bacterial growth and decay, attachment to and detachment from the surface, substrate utilization and disinfectant action involved in the model. The usefulness of the model is further enhanced by the incorporation of an expression for bulk reaction parameter relating it with the organic carbon. The model is validated and applied to study the sensitive behavior of the components using a hypothetical network. The developed model is able to simulate the biodegradable organic carbon threshold in accordance with the values reported in the literature. The spread of contaminant intruded into the system at any location can also be simulated by the model. The multicomponent model developed is useful for water supply authorities in identifying the locations with high substrate concentrations, bacterial growth and lower chlorine residuals. PMID:15087178

  6. Predicting liquid immiscibility in multicomponent nuclear waste glasses

    SciTech Connect

    Peeler, D.K.; Hrma, P.R.

    1994-04-01

    Taylor`s model for predicting amorphous phase separation in complex, multicomponent systems has been applied to high-level (simulated) radioactive waste glasses at the US Department of Energy`s Hanford site. Taylor`s model is primarily based on additions of modifying cations to a Na{sub 2}O-B{sub 2}O{sub 3}-SiO{sub 2} (NBS) submixture of the multicomponent glass. The position of the submixture relative to the miscibility dome defines the development probability of amorphous phase separation. Although prediction of amorphous phase separation in Hanford glasses (via experimental SEM/TEM analysis) is the primary thrust of this work; reported durability data is also provides limited insight into the composition/durability relationship. Using a modified model similar to Taylor`s, the results indicate that immiscibility may be predicted for multicomponent waste glasses by the addition of Li{sub 2}O to the ``alkali`` corner of the NBS submixture.

  7. Multi-Component Diffusion with Application To Computational Aerothermodynamics

    NASA Technical Reports Server (NTRS)

    Sutton, Kenneth; Gnoffo, Peter A.

    1998-01-01

    The accuracy and complexity of solving multicomponent gaseous diffusion using the detailed multicomponent equations, the Stefan-Maxwell equations, and two commonly used approximate equations have been examined in a two part study. Part I examined the equations in a basic study with specified inputs in which the results are applicable for many applications. Part II addressed the application of the equations in the Langley Aerothermodynamic Upwind Relaxation Algorithm (LAURA) computational code for high-speed entries in Earth's atmosphere. The results showed that the presented iterative scheme for solving the Stefan-Maxwell equations is an accurate and effective method as compared with solutions of the detailed equations. In general, good accuracy with the approximate equations cannot be guaranteed for a species or all species in a multi-component mixture. 'Corrected' forms of the approximate equations that ensured the diffusion mass fluxes sum to zero, as required, were more accurate than the uncorrected forms. Good accuracy, as compared with the Stefan- Maxwell results, were obtained with the 'corrected' approximate equations in defining the heating rates for the three Earth entries considered in Part II.

  8. Investigating the Structure of Multicomponent Gel-Phase Lipid Bilayers.

    PubMed

    Hartkamp, Remco; Moore, Timothy C; Iacovella, Christopher R; Thompson, Michael A; Bulsara, Pallav A; Moore, David J; McCabe, Clare

    2016-08-23

    Single- and multicomponent lipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), isostearyl isostearate, and heptadecanoyl heptadecanoate in the gel phase are studied via molecular dynamics simulations. It is shown that the structural properties of multicomponent bilayers can deviate strongly from the structures of their single-component counterparts. Specifically, the lipid mixtures are shown to adopt a compact packing by offsetting the positioning depths at which different lipid species are located in the bilayer. This packing mechanism affects the area per lipid, the bilayer height, and the chain tilt angles and has important consequences for other bilayer properties, such as interfacial hydrogen bonding and bilayer permeability. In particular, the simulations suggest that bilayers containing isostearyl isostearate or heptadecanoyl heptadecanoate are less permeable than pure 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine or DSPC bilayers. Furthermore, hydrogen-bond analysis shows that the residence times of lipid-water hydrogen bonds depend strongly on the bilayer composition, with longer residence times for bilayers that have a higher DSPC content. The findings illustrate and explain the fundamental differences between the properties of single- and multicomponent bilayers. PMID:27558724

  9. Hydrodynamic theory of diffusion in two-temperature multicomponent plasmas

    SciTech Connect

    Ramshaw, J.D.; Chang, C.H.

    1995-12-31

    Detailed numerical simulations of multicomponent plasmas require tractable expressions for species diffusion fluxes, which must be consistent with the given plasma current density J{sub q} to preserve local charge neutrality. The common situation in which J{sub q} = 0 is referred to as ambipolar diffusion. The use of formal kinetic theory in this context leads to results of formidable complexity. We derive simple tractable approximations for the diffusion fluxes in two-temperature multicomponent plasmas by means of a generalization of the hydrodynamical approach used by Maxwell, Stefan, Furry, and Williams. The resulting diffusion fluxes obey generalized Stefan-Maxwell equations that contain driving forces corresponding to ordinary, forced, pressure, and thermal diffusion. The ordinary diffusion fluxes are driven by gradients in pressure fractions rather than mole fractions. Simplifications due to the small electron mass are systematically exploited and lead to a general expression for the ambipolar electric field in the limit of infinite electrical conductivity. We present a self-consistent effective binary diffusion approximation for the diffusion fluxes. This approximation is well suited to numerical implementation and is currently in use in our LAVA computer code for simulating multicomponent thermal plasmas. Applications to date include a successful simulation of demixing effects in an argon-helium plasma jet, for which selected computational results are presented. Generalizations of the diffusion theory to finite electrical conductivity and nonzero magnetic field are currently in progress.

  10. Maltose metabolism of Pseudomonas fluorescens.

    PubMed Central

    Guffanti, A A; Corpe, W A

    1975-01-01

    Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis. PMID:240805

  11. Siderophore production by Pseudomonas pseudomallei.

    PubMed Central

    Yang, H M; Chaowagul, W; Sokol, P A

    1991-01-01

    Eighty-four strains of Pseudomonas pseudomallei isolated from patients with melioidosis were examined for siderophore production. All the strains were shown to produce siderophore both on chrome azurol S agar plates and in liquid medium under iron-deficient conditions. Chemical assays indicated that the siderophore belongs to the hydroxamate class. Addition of iron to the culture medium resulted in increased culture growth with markedly decreased yield of siderophore. Siderophore produced by strain U7 was purified by gel filtration chromatography, and the molecular weight was estimated to be 1,000. When this partially purified siderophore was added to culture medium, it promoted iron uptake by P. pseudomallei in the presence of EDTA and enhanced growth of the organism in the presence of transferrin. We have given this siderophore the trivial name malleobactin. PMID:1825486

  12. Glyphosate catabolism by Pseudomonas sp

    SciTech Connect

    Shinabarger, D.L.

    1986-01-01

    The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing (3-/sup 14/C) glyphosate revealed that approximately 50-59% of the C3 carbon was oxidized to CO/sub 2/. Fractionation of stationary phase cells labeled with (3-/sup 14/C)glyphosate revealed that from 45-47% of the assimilated C3 carbon is distributed to proteins and that amino acids methionine and serine are highly labeled. The nucleic acid bases adenine and guanine received 90% of the C3 label that was incorporated into nucleic acids, and the only pyrimidine base labeled was thymine. Pulse labeling of PG2982 cells with (3-/sup 14/C)glyphosate revealed that (3-/sup 14/C)sarcosine is an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of an enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. Phosphonate utilization by Pseudomonas sp. PG2982 was investigated. Each of the ten phosphonates tested were utilized as a sole source of phosphorus by PG2982. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. PG2982 cultures degraded phenylphosphonate to benzene and produced methane from methylphosphonate. The data indicate that PG2982 is capable of cleaving the carbon-phosphorus bond of several structurally different phosphonates.

  13. Antibiotic Conditioned Growth Medium of Pseudomonas Aeruginosa

    ERIC Educational Resources Information Center

    Benathen, Isaiah A.; Cazeau, Barbara; Joseph, Njeri

    2004-01-01

    A simple method to study the consequences of bacterial antibiosis after interspecific competition between microorganisms is presented. Common microorganisms are used as the test organisms and Pseudomonas aeruginosa are used as the source of the inhibitor agents.

  14. [Vasculitis caused by Pseudomonas: a case report].

    PubMed

    Escamilla, Y; Gutiérrez, M; Martínez, T; Bodoque, M; Gómez, J M; Moreno, A

    1996-01-01

    Pseudomona vasculitis is an exceptional disease. Only a few cases have been reported, non with oropharyngeal involvement. The case of a 30-year-old, HIV-positive man who suddenly developed septicemia and necrotizing lesions with tissue destruction of the oropharynx is reported. Histological study confirmed vasculitis. Pseudomona aeruginosa was isolated in peripheral blood and in the biopsy of the palatal lesion. Antibiotic treatment produced satisfactory results. PMID:8991411

  15. Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene.

    PubMed

    Haro, M A; de Lorenzo, V

    2001-02-13

    In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to

  16. Widespread distribution of soluble di-iron monooxygenase (SDIMO) genes in Arctic groundwater impacted by 1,4-dioxane.

    PubMed

    Li, Mengyan; Mathieu, Jacques; Yang, Yu; Fiorenza, Stephanie; Deng, Ye; He, Zhili; Zhou, Jizhong; Alvarez, Pedro J J

    2013-09-01

    Soluble di-iron monooxygenases (SDIMOs), especially group-5 SDIMOs (i.e., tetrahydrofuran and propane monooxygenases), are of significant interest due to their potential role in the initiation of 1,4-dioxane (dioxane) degradation. Functional gene array (i.e., GeoChip) analysis of Arctic groundwater exposed to dioxane since 1980s revealed that various dioxane-degrading SDIMO genes were widespread, and PCR-DGGE analysis showed that group-5 SDIMOs were present in every tested sample, including background groundwater with no known dioxane exposure history. A group-5 thmA-like gene was enriched (2.4-fold over background, p < 0.05) in source-zone samples with higher dioxane concentrations, suggesting selective pressure by dioxane. Microcosm assays with (14)C-labeled dioxane showed that the highest mineralization capacity (6.4 ± 0.1% (14)CO2 recovery during 15 days, representing over 60% of the amount degraded) corresponded to the source area, which was presumably more acclimated and contained a higher abundance of SDIMO genes. Dioxane mineralization ceased after 7 days and was resumed by adding acetate (0.24 mM) as an auxiliary substrate to replenish NADH, a key coenzyme for the functioning of monoxygenases. Acetylene inactivation tests further corroborated the vital role of monooxygenases in dioxane degradation. This is the first report of the prevalence of oxygenase genes that are likely involved in dioxane degradation and suggests their usefulness as biomarkers of dioxane natural attenuation. PMID:23909410

  17. Five monomeric hemocyanin subunits from Portunus trituberculatus: purification, spectroscopic characterization, and quantitative evaluation of phenol monooxygenase activity.

    PubMed

    Fujieda, Nobutaka; Yakiyama, Aki; Itoh, Shinobu

    2010-11-01

    Five kinds of monomeric subunits of arthropod hemocyanin have been isolated from swimming crab Portunus trituberculatus hemolymph. The copper centers holding a peroxo species, [(μ-η2:η2-peroxo)dicopper(II)], of these subunits exhibited almost the same UV-vis and visible region CD spectroscopic properties, indicating that they have a similar copper coordination geometry and an electronic structure. Under anaerobic conditions, the oxy-forms of the monomeric subunits were stable in 0.5 M borate buffer (pH 9.0) and reacted with 4-methylphenol (p-cresol) to show the phenolases (cresolase/phenol monooxygenase) activity in the presence of urea. To compare the phenolase (monooxygenase) reactivity, the reactivity of the isolated subunits has been examined quantitatively by using a simplified catalytic system, where the initial product catechol is trapped with borate anion of the buffer solution to prevent following catecholase reaction (Yamazaki and Itoh, 2003). The far-UV region CD spectra were measured in order to clarify the relationship between the content of the secondary structure and the phenolase reactivity. Even though the monomeric subunits exhibit a weak catalytic phenol monooxygenase activity, addition of urea (3 M) significantly enhances their catalytic activity. The differences of the phenolase activity among the monomeric subunits has been discussed on the basis of the spectroscopic analysis and reactivity studies in order to shed light on the enzymatic function of the arthropod hemocyanin in vivo. PMID:20727990

  18. OXIDATION OF POLYCHLORINATED BIPHENYLS BY PSEUDOMONAS SP. STRAIN LB400 AND PSEUDOMONAS PSEUDOALCALIGENES KF707

    EPA Science Inventory

    Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in th...

  19. A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism*

    PubMed Central

    Dresen, Carola; Lin, Leo Y.-C.; D'Angelo, Igor; Tocheva, Elitza I.; Strynadka, Natalie; Eltis, Lindsay D.

    2010-01-01

    Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent kcat/Km = 1000 ± 100 m−1 s−1 versus 700 ± 100 m−1 s−1). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent kcat/Km = 80 ± 40 m−1 s−1). In the presence of 3-HSA the Kmapp for O2 was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val367–Val394) could adopt two conformations differing by a rigid body rotation of 25° around Arg366. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids. PMID

  20. A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism.

    PubMed

    Dresen, Carola; Lin, Leo Y-C; D'Angelo, Igor; Tocheva, Elitza I; Strynadka, Natalie; Eltis, Lindsay D

    2010-07-16

    Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial

  1. Capsule production by Pseudomonas aeruginosa

    SciTech Connect

    Lynn, A.R.

    1984-01-01

    Mucoid strains of Pseudomonas aeruginosa, associated almost exclusively with chronic respiratory infections in patients with cystic fibrosis, possess a capsule composed of alginic acid similar to one produced by Azotobacter vinelandii. Recent reports have provided evidence that the biosynthetic pathway for alginate in P. aeruginosa may differ from the pathway proposed for A. vinelandii in that synthesis in P. aeruginosa may occur by way of the Entner-Doudoroff pathway. Incorporation of isotope from (6-/sup 14/C)glucose into alginate by both P. aueroginosa and A. vinelandii was 10-fold greater than that from either (1-/sup 14/C)/sup -/ or (2-/sup 14/C)glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate biosynthesis. These data strongly suggest that the Entner-Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii. The enzymes of carbohydrate metabolism in mucoid strains of P. aeruginosa appear to be unchanged whether alignate is actively produced or not and activities do not differ significantly from nonmucoid strain PAO.

  2. Isocyanide-based multicomponent reactions towards cyclic constrained peptidomimetics

    PubMed Central

    Koopmanschap, Gijs; Ruijter, Eelco

    2014-01-01

    Summary In the recent past, the design and synthesis of peptide mimics (peptidomimetics) has received much attention. This because they have shown in many cases enhanced pharmacological properties over their natural peptide analogues. In particular, the incorporation of cyclic constructs into peptides is of high interest as they reduce the flexibility of the peptide enhancing often affinity for a certain receptor. Moreover, these cyclic mimics force the molecule into a well-defined secondary structure. Constraint structural and conformational features are often found in biological active peptides. For the synthesis of cyclic constrained peptidomimetics usually a sequence of multiple reactions has been applied, which makes it difficult to easily introduce structural diversity necessary for fine tuning the biological activity. A promising approach to tackle this problem is the use of multicomponent reactions (MCRs), because they can introduce both structural diversity and molecular complexity in only one step. Among the MCRs, the isocyanide-based multicomponent reactions (IMCRs) are most relevant for the synthesis of peptidomimetics because they provide peptide-like products. However, these IMCRs usually give linear products and in order to obtain cyclic constrained peptidomimetics, the acyclic products have to be cyclized via additional cyclization strategies. This is possible via incorporation of bifunctional substrates into the initial IMCR. Examples of such bifunctional groups are N-protected amino acids, convertible isocyanides or MCR-components that bear an additional alkene, alkyne or azide moiety and can be cyclized via either a deprotection–cyclization strategy, a ring-closing metathesis, a 1,3-dipolar cycloaddition or even via a sequence of multiple multicomponent reactions. The sequential IMCR-cyclization reactions can afford small cyclic peptide mimics (ranging from four- to seven-membered rings), medium-sized cyclic constructs or peptidic macrocycles

  3. Multicomponent aerosol dynamics model UHMA: model development and validation

    NASA Astrophysics Data System (ADS)

    Korhonen, H.; Lehtinen, K. E. J.; Kulmala, M.

    2004-05-01

    A size-segregated aerosol dynamics model UHMA (University of Helsinki Multicomponent Aerosol model) was developed for studies of multicomponent tropospheric aerosol particles. The model includes major aerosol microphysical processes in the atmosphere with a focus on new particle formation and growth; thus it incorporates particle coagulation and multicomponent condensation, applying a revised treatment of condensation flux onto free molecular regime particles and the activation of nanosized clusters by organic vapours (Nano-Köhler theory), as well as recent parameterizations for binary H2SO4-H2O and ternary H2SO4-NH3-H2O homogeneous nucleation and dry deposition. The representation of particle size distribution can be chosen from three sectional methods: the hybrid method, the moving center method, and the retracking method in which moving sections are retracked to a fixed grid after a certain time interval. All these methods can treat particle emissions and atmospheric transport consistently, and are therefore suitable for use in large scale atmospheric models. In a test simulation against an accurate high resolution solution, all the methods showed reasonable treatment of new particle formation with 20 size sections although the hybrid and the retracking methods suffered from artificial widening of the distribution. The moving center approach, on the other hand, showed extra dents in the particle size distribution and failed to predict the onset of detectable particle formation. In a separate test simulation of an observed nucleation event, the model captured the key qualitative behaviour of the system well. Furthermore, its prediction of the organic volume fraction in newly formed particles, suggesting values as high as 0.5 for 3-4 nm particles and approximately 0.8 for 10 nm particles, agrees with recent indirect composition measurements.

  4. Hydrodynamic Theory of Atomic Mixing in Multicomponent Gases and Plasmas

    SciTech Connect

    Ramshaw, J D

    2001-08-22

    Atomic mixing in multicomponent gases and plasmas is usually described as a diffusional process. The diffusional description is an approximation to a more general dynamical description in which the motion of each individual species or material is governed by its own momentum equation, with appropriate coupling terms to represent the exchange of momentum between different species. These equations are not new, but they are scattered in the literature. Here we summarize the form of these species momentum equations, and the coupling coefficients therein, in sufficient detail to facilitate their inclusion and use to simulate atomic mixing in hydrodynamics codes.

  5. Physicochemical characteristics of the internal nitriding of multicomponent alloys

    SciTech Connect

    Petrova, L.G.

    1995-07-01

    Internal nitriding is the saturation of the deep layers of an alloy with nitrogen, which produces a structure composed of disperse nitride particles distributed in a solid solution. This distinguishes internal nitriding from conventional nitriding where a continuous nitride zone is formed in the surface layer. Therefore, internal nitrogenization, as in other processes of alloy saturation with implantation elements (e.g., internal oxidation), ensures the disperse strengthening of the alloy. The degree of strengthening is dependent on the amount and dispersion of the evolved nitrides and is associated with their thermodynamic stability and coagulation resistance. The study of the characteristics of internal nitriding of multicomponent alloys is of interest.

  6. Use of piezoelectric multicomponent force measuring devices in fluid mechanics

    NASA Technical Reports Server (NTRS)

    Richter, A.; Stefan, K.

    1979-01-01

    The characterisitics of piezoelectric multicomponent transducers are discussed, giving attention to the advantages of quartz over other materials. The main advantage of piezoelectric devices in aerodynamic studies is their ability to indicate rapid changes in the values of physical parameters. Problems in the accuracy of measurments by piezoelectric devices can be overcome by suitable design approaches. A practical example is given of how such can be utilized to measure rapid fluctuations of fluid forces exerted on a circular cylinder mounted in a water channel.

  7. Fluid description of multi-component solar partially ionized plasma

    SciTech Connect

    Khomenko, E. Collados, M.; Vitas, N.; Díaz, A.

    2014-09-15

    We derive self-consistent formalism for the description of multi-component partially ionized solar plasma, by means of the coupled equations for the charged and neutral components for an arbitrary number of chemical species, and the radiation field. All approximations and assumptions are carefully considered. Generalized Ohm's law is derived for the single-fluid and two-fluid formalism. Our approach is analytical with some order-of-magnitude support calculations. After general equations are developed, we particularize to some frequently considered cases as for the interaction of matter and radiation.

  8. Multicomponent modelling of Fischer-Tropsch slurry reactors

    SciTech Connect

    Van Vuuren, D.S.; Heydenrych, M.D.

    1985-08-01

    In the multicomponent model developed for a Fischer-Tropsch slurry reactor, the water-gas shift reaction is assumed to be in equilibrium. This is supported by literature data on iron-based catalysts above 523 K and synthesis gas conversion above about 50%. A Schulz-Flory product distribution is used. Investigation of the effects of back-mixing and interphase mass transfer using the model shows that, although the mass transfer rates in full-scale reactors are fast compared with reaction rates, the ratio of the mass transfer rates of reactants and products is important in determining gas velocity and gas hold-up and hence reactor performance.

  9. Dissipative shocks in multicomponent magneto rotating Lorentzian plasmas

    NASA Astrophysics Data System (ADS)

    Hussain, S.; Akhtar, N.; Hasnain, H.

    2015-11-01

    Nonlinear ion acoustic shocks in homogenous multicomponent electron-positron-ion (e-p-i) dissipative magneto rotating plasmas are studied. Dissipation in the plasma system is included via kinematic viscosity of ions. The electrons and positrons are Lorentzian and following kappa distribution function. Reductive perturbation technique is applied to derive Korteweg de Vries Burgers (KdVB) equation. The effects of variation of positron density, positron spectral index, temperature ratio of kappa distributed electrons to kappa distributed positrons, ion kinematic viscosity and rotational frequency effects are discussed in the context of pulsar magnetosphere.

  10. Margination Regimes and Drainage Transition in Confined Multicomponent Suspensions

    NASA Astrophysics Data System (ADS)

    Henríquez Rivera, Rafael G.; Sinha, Kushal; Graham, Michael D.

    2015-05-01

    A mechanistic theory is developed to describe segregation in confined multicomponent suspensions such as blood. It incorporates the two key phenomena arising in these systems at low Reynolds number: hydrodynamic pair collisions and wall-induced migration. In simple shear flow, several regimes of segregation arise, depending on the value of a "margination parameter" M . Most importantly, there is a critical value of M below which a sharp "drainage transition" occurs: one component is completely depleted from the bulk flow to the vicinity of the walls. Direct simulations also exhibit this transition as the size or flexibility ratio of the components changes.

  11. Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q

    SciTech Connect

    Shephard, E.A.; Fox, M.F.; Povey, S. ); Dolphin, C.T.; Phillips, I.R.; Smith, R. )

    1993-04-01

    The authors have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685--1689) to chromosome 1. They propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and 1. R. Phillips, 1991, J. Biol. Chem. 266: 12379--12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261--267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1 q23-q25. 28 refs., 3 figs., 2 tabs.

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

    PubMed Central

    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

  13. Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency.

    PubMed

    Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Rošker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

    2014-12-26

    Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. PMID:25361767

  14. Carbon Isotope Fractionations Associated with Methanotrophic Growth with the Soluble and Particulate Methane Monooxygenases

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.; Summons, Roger E.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Growth experiments with the RuMP-type methanotroph, Methylococcus capsulatus (Bath), have demonstrated that biomass and lipid biomarkers are significantly depleted in C-13 compared to the substrate methane and that the extent of fractionation is dependent on whether cells express the soluble (s) or particulate (p) methane monooxygenase (MMO). The presence or absence of the characteristic sMMO subunits was monitored using SDS-polyacrylamide gels. In M. capsulatus grown with no Cu supplementation, the characteristic sMMO subunits were observed in the soluble fraction throughout the entire growth period and biomass was depleted in C-13 by approximately 14,700 relative to substrate methane. In cells grown with 5uM Cu, no sMMO bands were observed and a greater fractionation of approximately 27,700 in resultant biomass was obtained. Methanol growth experiments with M. capsulatus and with a RuMP methylotroph, Methylophilus methylotrophus, in which biomass measurements yielded depletions in C-13 of 9 and 5%(sub o), respectively, suggest that oxidation of methane is the major fractionation step. Growth of M. capsulatus at a low level of oxygen, approximately 0.5%, had no significant effect on carbon isotope fractionation by either sMMO or pMMO. These observations are significant for identification of molecular biomarkers; and methanotrophic contributions to carbon isotope composition in natural environments.

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

    PubMed Central

    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

  16. Chopping and Changing: the Evolution of the Flavin-dependent Monooxygenases.

    PubMed

    Mascotti, Maria Laura; Juri Ayub, Maximiliano; Furnham, Nicholas; Thornton, Janet M; Laskowski, Roman A

    2016-07-31

    Flavin-dependent monooxygenases play a variety of key physiological roles and are also very powerful biotechnological tools. These enzymes have been classified into eight different classes (A-H) based on their sequences and biochemical features. By combining structural and sequence analysis, and phylogenetic inference, we have explored the evolutionary history of classes A, B, E, F, and G and demonstrate that their multidomain architectures reflect their phylogenetic relationships, suggesting that the main evolutionary steps in their divergence are likely to have arisen from the recruitment of different domains. Additionally, the functional divergence within in each class appears to have been the result of other mechanisms such as a complex set of single-point mutations. Our results reinforce the idea that a main constraint on the evolution of cofactor-dependent enzymes is the functional binding of the cofactor. Additionally, a remarkable feature of this family is that the sequence of the key flavin adenine dinucleotide-binding domain is split into at least two parts in all classes studied here. We propose a complex set of evolutionary events that gave rise to the origin of the different classes within this family. PMID:27423402

  17. Immobilization of P450 monooxygenase and chloroplast for use in light-driven bioreactors.

    PubMed

    Hara, M; Iazvovskaia, S; Ohkawa, H; Asada, Y; Miyake, J

    1999-01-01

    P450 monooxygenases exhibit great potential for application to bioreactors for the decomposition of various hydrophobic chemicals including pollutant compounds. P450-containing microsomes were immobilized in spinach chloroplasts for use in light-driven bioreactors. We tested three methods (entrapment, adsorption and cross-linking) to immobilize chloroplasts and yeast microsomes containing a genetically engineered fusion enzyme between rat P450 1A1 and yeast P450 reductase. Entrapment in agarose gave the best activity for the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin under illumination of 6200 lx. We then tested three light-driven bioreactors (two column-type and one batch-type reactors developed) using the immobilized gels. A two-phase column-type reactor with separately immobilized microsomes and chloroplasts showed a higher conversion rate than a reactor with co-immobilization of both components. The reactor showed a turnover rate of 6.32 mol product/mol P450/min after a 40-min run, and 2.49 after a 180-min run. These turnover rates are higher than the values reported by others using immobilized microsomal P450s. PMID:16232556

  18. Structure of T4moF, the Toluene 4-Monooxygenase Ferredoxin Oxidoreductase.

    PubMed

    Acheson, Justin F; Moseson, Hannah; Fox, Brian G

    2015-09-29

    The 1.6 Å crystal structure of toluene 4-monooxygenase reductase T4moF is reported. The structure includes ferredoxin, flavin, and NADH binding domains. The position of the ferredoxin domain relative to the other two domains represents a new configuration for the iron-sulfur flavoprotein family. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a plausible pathway for electron transfer between the redox cofactors. Energy-minimized docking of NADH and calculation of hingelike motions between domains suggest how simple coordinated shifts of residues at the C-terminus of the enzyme could expose the N5 position of FAD for productive interaction with the nicotinamide ring. The domain configuration revealed by the T4moF structure provides an excellent steric and electrostatic match to the obligate electron acceptor, Rieske-type [2Fe-2S] ferredoxin T4moC. Protein-protein docking and energy minimization of the T4moFC complex indicate that T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface. PMID:26309236

  19. (+)-Abscisic Acid 8′-Hydroxylase Is a Cytochrome P450 Monooxygenase1

    PubMed Central

    Krochko, Joan E.; Abrams, Garth D.; Loewen, Mary K.; Abrams, Suzanne R.; Cutler, Adrian J.

    1998-01-01

    Abscisic acid (ABA) 8′-hydroxylase catalyzes the first step in the oxidative degradation of (+)-ABA. The development of a robust in vitro assay has now permitted detailed examination and characterization of this enzyme. Although several factors (buffer, cofactor, and source tissue) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevich [1997] J Exp Bot 48: 1787–1795). (+)-ABA 8′-hydroxylase is an integral membrane protein that is localized to the microsomal fraction in suspension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, although there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)-ABA was progressively inhibited at O2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N2. (+)-ABA 8′-hydroxylase activity was inhibited by tetcyclacis (50% inhibition at 10−6 m), cytochrome c (oxidized form), and CO. The CO inhibition was reversible by light from several regions of the visible spectrum, but most efficiently by blue and amber light. These data strongly support the contention that (+)-ABA 8′-hydroxylase is a cytochrome P450 monooxygenase. PMID:9808729

  20. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway.

    PubMed

    Umemoto, Naoyuki; Nakayasu, Masaru; Ohyama, Kiyoshi; Yotsu-Yamashita, Mari; Mizutani, Masaharu; Seki, Hikaru; Saito, Kazuki; Muranaka, Toshiya

    2016-08-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  1. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    PubMed

    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. PMID:25296660

  2. Lead discovery for human kynurenine 3-monooxygenase by high-throughput RapidFire mass spectrometry.

    PubMed

    Lowe, Denise M; Gee, Michelle; Haslam, Carl; Leavens, Bill; Christodoulou, Erica; Hissey, Paul; Hardwicke, Philip; Argyrou, Argyrides; Webster, Scott P; Mole, Damian J; Wilson, Kris; Binnie, Margaret; Yard, Beverley A; Dean, Tony; Liddle, John; Uings, Iain; Hutchinson, Jonathan P

    2014-04-01

    Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z' value 0.8) and provided several tractable hit series for further investigation. PMID:24381207

  3. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

    PubMed Central

    Hirani, Nisha; Westenberg, Marcel; Seed, Paul T.; Petalcorin, Mark I. R.; Dolphin, Colin T.

    2016-01-01

    ABSTRACT Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation. PMID:27010030

  4. Suppressed expression of choline monooxygenase in sugar beet on the accumulation of glycine betaine.

    PubMed

    Yamada, Nana; Takahashi, Hiroyuki; Kitou, Kunihide; Sahashi, Kosuke; Tamagake, Hideto; Tanaka, Yoshito; Takabe, Teruhiro

    2015-11-01

    Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of choline. Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation. PMID:26302482

  5. Regulation of betaine synthesis by precursor supply and choline monooxygenase expression in Amaranthus tricolor.

    PubMed

    Bhuiyan, Nazmul H; Hamada, Akira; Yamada, Nana; Rai, Vandna; Hibino, Takashi; Takabe, Teruhiro

    2007-01-01

    In plants, betaine is synthesized upon abiotic stress via choline oxidation, in which choline monooxygenase (CMO) is a key enzyme. Although it had been thought that betaine synthesis is well regulated to protect abiotic stress, it is shown here that an exogenous supply of precursors such as choline, serine, and glycine in the betaine-accumulating plant Amaranthus tricolor further enhances the accumulation of betaine under salt stress, but not under normal conditions. Addition of isonicotinic acid hydrazide, an inhibitor of glycine decarboxylase, inhibited the salinity-induced accumulation of betaine. Salt-induced accumulation of A. tricolor CMO (AmCMO) and betaine was much slower in roots than in leaves, and a transient accumulation of proline was observed in the roots. Antisense expression of AmCMO mRNA suppressed the salt-induced accumulation of AmCMO and betaine, but increased the level of choline approximately 2- 3-fold. This indicates that betaine synthesis is highly regulated by AmCMO expression. The genomic DNA, including the upstream region (1.6 kbp), of AmCMO was isolated. Deletion analysis of the AmCMO promoter region revealed that the 410 bp fragment upstream of the translation start codon contains the sequence responsive to salt stress. These data reveal that the promoter sequence of CMO, in addition to precursor supply, is important for the accumulation of betaine in the betaine-accumulating plant A. tricolor. PMID:18182425

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

    PubMed

    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. PMID:26752518

  7. Yeast flavin-containing monooxygenase is induced by the unfolded protein response

    PubMed Central

    Suh, Jung-Keun; Robertus, Jon D.

    2000-01-01

    Flavin-containing monooxygenase from yeast (yFMO) carries out the O2- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide. FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40%. The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell. However, yFMO is vital to the yeast response to reductive stress. FMO1 deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type. The FMO1 gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response. Reductive stress can increase yFMO activity at least 6-fold. This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids. The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents. PMID:10618381

  8. Molecular and dietary regulation of beta,beta-carotene 15,15'-monooxygenase 1 (BCMO1).

    PubMed

    Lietz, Georg; Lange, Jennifer; Rimbach, Gerald

    2010-10-01

    beta,beta-Carotene 15,15'-monooxygenase-1 (BCMO1) is a key enzyme in vitamin A metabolism in mammals. Various dietary components such as non-pro-vitamin A carotenoids, fat, and polyphenols have been shown to influence the intestinal absorption and conversion of pro-vitamin A carotenoids. Furthermore, vitamin A deficiency has been shown to induce BCMO1 expression, whereas supplementation with vitamin A or its active metabolites, all-trans and 9-cis retinoic acid, dose-dependently reverse these effects. A diet-responsive regulatory network involving the intestine specific homeodomain transcription factor ISX has been shown to regulate the intestinal vitamin A uptake and production via a negative feedback control. Furthermore, non-synonymous single nucleotide polymorphisms in the human BCMO1 gene have been discovered causing observably reduced BCMO1 activity. Detailed knowledge about BCMO1 regulation as well as genetic variations causing variable cleavage activities may provide a background, on which individual and/or population based dietary recommendations for beta-carotene and vitamin A intake could be established. PMID:20599666

  9. A C4-oxidizing Lytic Polysaccharide Monooxygenase Cleaving Both Cellulose and Cello-oligosaccharides*

    PubMed Central

    Isaksen, Trine; Westereng, Bjørge; Aachmann, Finn L.; Agger, Jane W.; Kracher, Daniel; Kittl, Roman; Ludwig, Roland; Haltrich, Dietmar; Eijsink, Vincent G. H.; Horn, Svein J.

    2014-01-01

    Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. PMID:24324265

  10. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

    PubMed

    Isaksen, Trine; Westereng, Bjørge; Aachmann, Finn L; Agger, Jane W; Kracher, Daniel; Kittl, Roman; Ludwig, Roland; Haltrich, Dietmar; Eijsink, Vincent G H; Horn, Svein J

    2014-01-31

    Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. PMID:24324265

  11. Discovery and characterization of a new family of lytic polysaccharide mono-oxygenases

    PubMed Central

    Hemsworth, Glyn R.; Henrissat, Bernard; Davies, Gideon J.; Walton, Paul H.

    2014-01-01

    Lytic polysaccharide mono-oxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They currently attract much attention due to their potential use in biomass conversion, notably in the production of biofuels. Past work has identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the 3-D structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active centre featuring the histidine brace active site, but is distinct in terms of its active site details and its EPR spectroscopy. The new AA11 family expands the LPMO clan with the potential to broaden both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs. PMID:24362702

  12. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay

    PubMed Central

    2012-01-01

    Background Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Results Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. Conclusions P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components. PMID:23102010

  13. Novel hydrocarbon monooxygenase genes in the metatranscriptome of a natural deep-sea hydrocarbon plume.

    PubMed

    Li, Meng; Jain, Sunit; Baker, Brett J; Taylor, Chris; Dick, Gregory J

    2014-01-01

    Particulate membrane-associated hydrocarbon monooxygenases (pHMOs) are critical components of the aerobic degradation pathway for low molecular weight hydrocarbons, including the potent greenhouse gas methane. Here, we analysed pHMO gene diversity in metagenomes and metatranscriptomes of hydrocarbon-rich hydrothermal plumes in the Guaymas Basin (GB) and nearby background waters in the deep Gulf of California. Seven distinct phylogenetic groups of pHMO were present and transcriptionally active in both plume and background waters, including several that are undetectable with currently available polymerase chain reaction (PCR) primers. The seven groups of pHMOs included those related to a putative ethane oxidizing Methylococcaceae-like group, a group of the SAR324 Deltaproteobacteria, three deep-sea clades (Deep sea-1/symbiont-like, Deep sea-2/PS-80 and Deep sea-3/OPU3) within gammaproteobacterial methanotrophs, one clade related to Group Z and one unknown group. Differential abundance of pHMO gene transcripts in plume and background suggests niche differentiation between groups. Corresponding 16S rRNA genes reflected similar phylogenetic and transcriptomic abundance trends. The novelty of transcriptionally active pHMOs we recovered from a hydrocarbon-rich hydrothermal plume suggests there are significant gaps in our knowledge of the diversity and function of these enzymes in the environment. PMID:23826624

  14. Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic Insights

    PubMed Central

    Alqassim, Saif S.; Urquiza, Mauricio; Borgnia, Eitan; Nagib, Marc; Amzel, L. Mario; Bianchet, Mario A.

    2016-01-01

    MICALs (Molecule Interacting with CasL) are conserved multidomain enzymes essential for cytoskeletal reorganization in nerve development, endocytosis, and apoptosis. In these enzymes, a type-2 calponin homology (CH) domain always follows an N-terminal monooxygenase (MO) domain. Although the CH domain is required for MICAL-1 cellular localization and actin-associated function, its contribution to the modulation of MICAL activity towards actin remains unclear. Here, we present the structure of a fragment of MICAL-1 containing the MO and the CH domains—determined by X-ray crystallography and small angle scattering—as well as kinetics experiments designed to probe the contribution of the CH domain to the actin-modification activity. Our results suggest that the CH domain, which is loosely connected to the MO domain by a flexible linker and is far away from the catalytic site, couples F-actin to the enhancement of redox activity of MICALMO-CH by a cooperative mechanism involving a trans interaction between adjacently bound molecules. Binding cooperativity is also observed in other proteins regulating actin assembly/disassembly dynamics, such as ADF/Cofilins. PMID:26935886

  15. Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

    PubMed Central

    Smith, Stephen M.; Rawat, Swati; Telser, Joshua; Hoffman, Brian M.; Stemmler, Timothy L.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of CuI and CuII, of which the latter exhibits two distinct type 2 CuII electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu–O/N and Cu–Cu ligand environments with a Cu–Cu interaction at 2.52–2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models. PMID:22013879

  16. Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium.

    PubMed

    Nielsen, A K; Gerdes, K; Murrell, J C

    1997-07-01

    The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO). These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane-bound or particulate form (pMMO). Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper-biomass ratios, whereas pMMO is expressed at high copper-biomass ratios. In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions. Data suggest that transcription of the M. trichosporium OB3b sMMO gene cluster is directed from a sigma54-like and a sigma70-like promoter. The pMMO (pmo) genes of M. capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb. The synthesis of this mRNA was activated by copper ions. Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs. This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression. PMID:9282751

  17. Oxidation of ultrafast radical clock substrate probes by the soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Valentine, A M; LeTadic-Biadatti, M H; Toy, P H; Newcomb, M; Lippard, S J

    1999-04-16

    Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling. PMID:10196150

  18. Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity.

    PubMed

    Mitchell, Martin J; Brescia, Aaron I; Smith, Stan L; Morgan, E David

    2007-09-01

    The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M. PMID:17694563

  19. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis.

    PubMed

    Shih, Diana M; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J Mark; Vallim, Thomas; Bennett, Brian J; Graham, Mark; Hazen, Stanley L; Lusis, Aldons J

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  20. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis[S

    PubMed Central

    Shih, Diana M.; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J. Mark; Vallim, Thomas; Bennett, Brian J.; Graham, Mark; Hazen, Stanley L.; Lusis, Aldons J.

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  1. Crystallization and preliminary structural analysis of dibenzothiophene monooxygenase (DszC) from Rhodococcus erythropolis

    PubMed Central

    Duan, Xiaolu; Zhang, Liang; Zhou, Daming; Ji, Kaihua; Ma, Ting; Shui, Wenqing; Li, Guoqiang; Li, Xin

    2013-01-01

    Dibenzothiophene (DBT) and its derivatives are typical sulfur compounds found in fossil fuels. These compounds show resistance to the hydrodesulfuriz­ation treatment that is commonly used in industry. Dibenzothiophene monooxygenase (DszC) is responsible for the oxidation of DBT, which is the first and the rate-limiting step in the DBT enzymatic desulfurization 4S pathway. In this study, the crystal structure of DszC from Rhodococcus erythropolis DS-3 is reported. The crystal of native DszC belonged to space group P1, with unit-cell parameters a = 96.16, b = 96.27, c = 98.56 Å, α = 81.03, β = 67.57, γ = 85.84°. To determine the phase, SAD X-ray diffraction data were collected from a SeMet-derivative DszC crystal, which also belonged to space group P1, with unit-cell parameters a = 95.379, b = 95.167, c = 94.891 Å, α = 87.046, β = 70.536, γ = 79.738°. Further structural analysis of DszC is in progress. PMID:23722833

  2. Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase.

    PubMed

    Jennewein, S; Rithner, C D; Williams, R M; Croteau, R B

    2001-11-20

    A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species. PMID:11707604

  3. Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro.

    PubMed

    Brodie, M J; Boobis, A R; Bulpitt, C J; Davies, D S

    1981-01-01

    The effects of liver disease and environmental factors on hepatic microsomal cytochrome P-450 content, NADPH-cytochrome c reductase (reductase) activity and aryl hydrocarbon hydroxylase (AHH) activity have been simultaneously investigated in 70 patients undergoing diagnostic liver biopsy. The activity of reductase was not significantly affected by the presence of liver disease or any of the environmental factors studied. Cytochrome P-450 content decreased with increasing severity of liver disease whereas AHH activity was only significantly reduced in biopsies showing hepatocellular destruction. None of the parameters of monooxygenase activity varied significantly with the age or sex of the patients. Alcohol excess was associated with decreased cytochrome P-450 content and AHH activity and this effect was independent of the histological status of the biopsy. Both high caffeine intake and cigarette smoking increased AHH activity in the absence of any change in cytochrome P-450 content. There was a positive correlation between the number of meat meals eaten per week and cytochrome P-450 content. Chronic treatment with enzyme-inducing anticonvulsants appeared to increase both cytochrome P-450 content and AHH activity. Despite differential effects of liver disease and environmental influences on cytochrome P-450 content and AHH activity there was a highly significant correlation between the two parameters. The results of the present study correlate well with the known effects of disease and environment on drug metabolism in vivo. PMID:7308271

  4. Monooxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase.

    PubMed

    Bui, Q D; Weisz, J

    1989-02-01

    Microsomes from rat anterior pituitaries (AP) were incubated with (3H)estradiol under conditions previously shown to support catecholestrogen (CE) formation by placental microsomes via an NADPH- or an organic hydroperoxide-dependent, peroxidatic mechanism. Under conditions optimized for monooxygenase activity (pH 8.0, 5 mM NADPH), 4-hydroxylation predominated (apparent Vmax = 65 pmol and 13 pmol/mg protein/30 min for 4- and 2-hydroxy-E2, respectively). Under conditions optimized for peroxidatic activity (pH 6.0, 50 mM cumene hydroperoxide) 2- and 4-hydroxylated-E2 were produced in similar amounts. Thus in the AP, unlike in other target tissues studied, NADPH-dependent CE synthetase is a 4-hydroxylase and significant 2-hydroxylation occurs only via the peroxidatic mechanism. We propose that 4-hydroxylated CEs, which are both potent, long acting estrogens and catechols, serve as local mediators of actions of phenolic estrogens on the AP. PMID:2536311

  5. Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes.

    PubMed

    Boyd, Derek R; Sharma, Narain D; McMurray, Brian; Haughey, Simon A; Allen, Christopher C R; Hamilton, John T G; McRoberts, W Colin; O'Ferrall, Rory A More; Nikodinovic-Runic, Jasmina; Coulombel, Lydie A; O'Connor, Kevin E

    2012-01-28

    Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b]thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b]thiophene sulfoxide and 2-methyl benzo[b]thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b]thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b]thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring. PMID:22134441

  6. Evidence for oxygen binding at the active site of particulate methane monooxygenase.

    PubMed

    Culpepper, Megen A; Cutsail, George E; Hoffman, Brian M; Rosenzweig, Amy C

    2012-05-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α(3)β(3)γ(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η(2):η(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site. PMID:22540911

  7. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M.

    PubMed

    Smith, Stephen M; Rawat, Swati; Telser, Joshua; Hoffman, Brian M; Stemmler, Timothy L; Rosenzweig, Amy C

    2011-11-29

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models. PMID:22013879

  8. Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene

    SciTech Connect

    Hsienchyang Tsien; Hanson, R.S. )

    1992-03-01

    Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Baath also produces a soluble MMO.

  9. Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

    SciTech Connect

    Smith, Stephen M.; Rawat, Swati; Telser, Joshua; Hoffman, Brian M.; Stemmler, Timothy L.; Rosenzweig, Amy C.

    2012-02-08

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain {approx}2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu{sup I} and Cu{sup II}, of which the latter exhibits two distinct type 2 Cu{sup II} electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 {angstrom}. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 {angstrom} resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

  10. Bacteriohemerythrin bolsters the activity of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath).

    PubMed

    Chen, Kelvin H-C; Wu, Hsin-Hui; Ke, Si-Fu; Rao, Ya-Ting; Tu, Chia-Ming; Chen, Yu-Ping; Kuei, Kuo-Hsuan; Chen, Ying-Siao; Wang, Vincent C-C; Kao, Wei-Chun; Chan, Sunney I

    2012-06-01

    Recently, a native bacteriohemerythrin (McHr) has been identified in Methylococcus capsulatus (Bath). Both the particulate methane monooxygenase (pMMO) and McHr are over-expressed in cells of this bacterium when this strain of methanotroph is cultured and grown under high copper to biomass conditions. It has been suggested that the role of the McHr is to provide a shuttle to transport dioxygen from the cytoplasm of the cell to the intra-cytoplasmic membranes for consumption by the pMMO. Indeed, McHr enhances the activity of the pMMO when pMMO-enriched membranes are used to assay the enzyme activity. We find that McHr can dramatically improve the activity of pMMO toward the epoxidation of propylene to propylene oxide. The maximum activity is observed at a pMMO to McHr concentration ratio of 4:1, where we have obtained specific activities of 103.7nmol propylene oxide/min/mg protein and 122.8nmol propylene oxide/min/mg protein at 45°C when the turnover is driven by NADH and duroquinol, respectively. These results are consistent with the suggestion that the bacterium requires McHr to deliver dioxygen to the pMMO in the intra-cytoplasmic membranes to accomplish efficient catalysis of methane oxidation when the enzyme is over-expressed in the cells. PMID:22484247

  11. Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants.

    PubMed

    Hibino, Takashi; Waditee, Rungaroon; Araki, Etsuko; Ishikawa, Hiroshi; Aoki, Kenji; Tanaka, Yoshito; Takabe, Teruhiro

    2002-11-01

    In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO). Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine non-accumulating plant. Here, we examined the functional properties of CMO expressed in Escherichia coli, cyanobacterium, and Arabidopsis thaliana. We found that E. coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDH-deleted E. coli mutant. Changes of Cys-181 in spinach CMO to Ser, Thr, and Ala and His-287 to Gly, Val, and Ala abolished the accumulation of betaine. The Arabidopsis CMO-like gene was transcribed in Arabidopsis, but its protein was not detected. When the Arabidopsis CMO-like gene was expressed in E. coli, the protein was detected but was found not to promote betaine sysnthesis. Overexpression of spinach CMO in E. coli, Synechococcus sp. PCC7942, and Arabidopsis conferred resistance to abiotic stress. These facts clearly indicate that CMO, but not the CMO-like protein, could oxidize choline and that Cys-181 and His-287 are involved in the binding of Fe-S cluster and Fe, respectively. PMID:12192001

  12. Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics.

    PubMed

    David, J P; Boyer, S; Mesneau, A; Ball, A; Ranson, H; Dauphin-Villemant, C

    2006-05-01

    The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed. PMID:16651188

  13. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress.

    PubMed

    Hirani, Nisha; Westenberg, Marcel; Seed, Paul T; Petalcorin, Mark I R; Dolphin, Colin T

    2016-01-01

    Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney - an appropriate site if it too is, or once was, involved in osmoregulation. PMID:27010030

  14. Reaction Mechanism of the Bicopper Enzyme Peptidylglycine α-Hydroxylating Monooxygenase*

    PubMed Central

    Abad, Enrique; Rommel, Judith B.; Kästner, Johannes

    2014-01-01

    Peptidylglycine α-hydroxylating monooxygenase is a noninteracting bicopper enzyme that stereospecifically hydroxylates the terminal glycine of small peptides for its later amidation. Neuroendocrine messengers, such as oxytocin, rely on the biological activity of this enzyme. Each catalytic turnover requires one oxygen molecule, two protons from the solvent, and two electrons. Despite this enzyme having been widely studied, a consensus on the reaction mechanism has not yet been found. Experiments and theoretical studies favor a pro-S abstraction of a hydrogen atom followed by the rebinding of an OH group. However, several hydrogen-abstracting species have been postulated; because two protons are consumed during the reaction, several protonation states are available. An electron transfer between the copper atoms could play a crucial role for the catalysis as well. This leads to six possible abstracting species. In this study, we compare them on equal footing. We perform quantum mechanics/molecular mechanics calculations, considering the glycine hydrogen abstraction. Our results suggest that the most likely mechanism is a protonation of the abstracting species before the hydrogen abstraction and another protonation as well as a reduction before OH rebinding. PMID:24668808

  15. Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase.

    PubMed

    Courtade, Gaston; Wimmer, Reinhard; Røhr, Åsmund K; Preims, Marita; Felice, Alfons K G; Dimarogona, Maria; Vaaje-Kolstad, Gustav; Sørlie, Morten; Sandgren, Mats; Ludwig, Roland; Eijsink, Vincent G H; Aachmann, Finn Lillelund

    2016-05-24

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action. PMID:27152023

  16. Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency*

    PubMed Central

    Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Rošker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

    2014-01-01

    Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. PMID:25361767

  17. (2+1)-dimensional non-isospectral multi-component AKNS equations and its integrable couplings

    SciTech Connect

    Sun Yepeng

    2010-03-08

    (2+1)-dimensional non-isospectral multi-component AKNS equations are derived from an arbitrary order matrix spectral problem. As a reduction, (2+1)-dimensional non-isospectral multi-component Schroedinger equations are obtained. Moreover, new (2+1)-dimensional non-isospectral integrable couplings of the resulting AKNS equations are constructed by enlarging the associated matrix spectral problem.

  18. An Evaluation of a Multicomponent Early Literacy Program for Students with Severe Developmental Disabilities

    ERIC Educational Resources Information Center

    Browder, Diane; Ahlgrim-Delzell, Lynn; Flowers, Claudia; Baker, Joshua

    2012-01-01

    This study evaluated the effectiveness of a multicomponent early literacy curriculum that included phonics and phonemic awareness in comparison to a sight word approach. A total of 93 students with severe developmental disabilities who were enrolled in Grades K through 4 were randomly assigned to either a multicomponent early literacy curriculum…

  19. Alcohol-Related Information in Multi-Component Interventions and College Students' Drinking Behavior

    ERIC Educational Resources Information Center

    Thadani, Vandana; Huchting, Karen; LaBrie, Joseph

    2009-01-01

    Education-only interventions produce little change in drinking behaviors; but, multi-component prevention programs, which include alcohol information as one feature, can decrease drinking. This study examined the role of alcohol knowledge in a multi-component intervention previously found to reduce first-year female college students' alcohol…

  20. Catalysis of Cascade and Multicomponent Reactions of Carbonyl Compounds and CH Acids by Electricity.

    PubMed

    Elinson, Michail N; Vereshchagin, Anatoly N; Ryzhkov, Fedor V

    2016-08-01

    This review is concerned with modern trends in the use of electrochemically induced chain reactions in cascade and multicomponent electroorganic synthesis. The review summarizes the data on the use of electrochemically induced chain reactions in cascade and multicomponent organic synthesis, which were published mainly in the last decade. PMID:27276501

  1. Glycerophospholipid synthesis and functions in Pseudomonas.

    PubMed

    Kondakova, Tatiana; D'Heygère, François; Feuilloley, Marc J; Orange, Nicole; Heipieper, Hermann J; Duclairoir Poc, Cécile

    2015-09-01

    The genus Pseudomonas is one of the most heterogeneous groups of eubacteria, presents in all major natural environments and in wide range of associations with plants and animals. The wide distribution of these bacteria is due to the use of specific mechanisms to adapt to environmental modifications. Generally, bacterial adaptation is only considered under the aspect of genes and protein expression, but lipids also play a pivotal role in bacterial functioning and homeostasis. This review resumes the mechanisms and regulations of pseudomonal glycerophospholipid synthesis, and the roles of glycerophospholipids in bacterial metabolism and homeostasis. Recently discovered specific pathways of P. aeruginosa lipid synthesis indicate the lineage dependent mechanisms of fatty acids homeostasis. Pseudomonas glycerophospholipids ensure structure functions and play important roles in bacterial adaptation to environmental modifications. The lipidome of Pseudomonas contains a typical eukaryotic glycerophospholipid--phosphatidylcholine -, which is involved in bacteria-host interactions. The ability of Pseudomonas to exploit eukaryotic lipids shows specific and original strategies developed by these microorganisms to succeed in their infectious process. All compiled data provide the demonstration of the importance of studying the Pseudomonas lipidome to inhibit the infectious potential of these highly versatile germs. PMID:26148574

  2. Pseudomonas aeruginosa biofilms in disease.

    PubMed

    Mulcahy, Lawrence R; Isabella, Vincent M; Lewis, Kim

    2014-07-01

    Pseudomonas aeruginosa is a ubiquitous organism that is the focus of intense research because of its prominent role in disease. Due to its relatively large genome and flexible metabolic capabilities, this organism exploits numerous environmental niches. It is an opportunistic pathogen that sets upon the human host when the normal immune defenses are disabled. Its deadliness is most apparent in cystic fibrosis patients, but it also is a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disorder, surface growth on implanted biomaterials, and within hospital surface and water supplies, where it poses a host of threats to vulnerable patients (Peleg and Hooper, N Engl J Med 362:1804-1813, 2010; Breathnach et al., J Hosp Infect 82:19-24, 2012). Once established in the patient, P. aeruginosa can be especially difficult to treat. The genome encodes a host of resistance genes, including multidrug efflux pumps (Poole, J Mol Microbiol Biotechnol 3:255-264, 2001) and enzymes conferring resistance to beta-lactam and aminoglycoside antibotics (Vahdani et al., Annal Burns Fire Disast 25:78-81, 2012), making therapy against this gram-negative pathogen particularly challenging due to the lack of novel antimicrobial therapeutics (Lewis, Nature 485: 439-440, 2012). This challenge is compounded by the ability of P. aeruginosa to grow in a biofilm, which may enhance its ability to cause infections by protecting bacteria from host defenses and chemotherapy. Here, we review recent studies of P. aeruginosa biofilms with a focus on how this unique mode of growth contributes to its ability to cause recalcitrant infections. PMID:24096885

  3. New naphthalene-degrading marine Pseudomonas strains.

    PubMed Central

    García-Valdés, E; Cozar, E; Rotger, R; Lalucat, J; Ursing, J

    1988-01-01

    Over 100 strains that utilized naphthalene as the only carbon and energy source were isolated from samples of marine sediments taken from a heavily polluted area. The isolates were characterized taxonomically and physiologically. Most of these strains belonged to the genus Pseudomonas, and seven of them did not fit any previous taxonomic description. They differed from type strains in a few biochemical characteristics and in the utilization of aromatic compounds. None had catechol 1,2-dioxygenase activity, and catechol 2,3-dioxygenase was responsible for the aromatic ring cleavage. DNA hybridization demonstrated a close relationship between two isolates and the Pseudomonas stutzeri type strain, and between five isolates and the Pseudomonas testosteroni type strain. On the basis of nutritional and enzymatic characteristics, it was assumed that the seven isolates represent new biovars belonging to the species P. testosteroni and P. stutzeri that are able to degrade aromatic hydrocarbons. Images PMID:3202629

  4. New naphthalene-degrading marine Pseudomonas strains

    SciTech Connect

    Garcia-Valdes, E.; Cozar, E.; Rotger, R. Lalucat, J. ); Ursing, J. )

    1988-10-01

    Over 100 strains that utilized naphthalene as the only carbon and energy source were isolated from samples of marine sediments taken from a heavily polluted area. The isolates were characterized taxonomically and physiologically. Most of these strains belonged to the genus Pseudomonas, and seven of them did not fit any previous taxonomic description. They differed from type strains in a few biochemical characteristics and in the utilization of aromatic compounds. None had catechol 1,2-dioxygenase activity, and catechol 2,3-dioxygenase was responsible for the aromatic ring cleavage. DNA hybridizations demonstrated a close relationship between two isolates and the Pseudomonas stutzeri type strain, and between five isolates and the Pseudomonas testosteroni type strain. On the basis of nutritional and enzymatic characteristics, it was assumed that the seven isolates represent new biovars belonging to the species P. testosteroni and P. stutzeri that are able to degrade aromatic hydrocarbons.

  5. Lateral Organization of Lipids in Multi-component Liposomes

    NASA Astrophysics Data System (ADS)

    Ramachandran, Sanoop; Laradji, Mohamed; Sunil Kumar, P. B.

    2009-04-01

    Inspite of the fluid nature and low elastic modulus, membranes play a crucial role in maintaining the structural integrity of the cell. Recent experiments have challenged the passive nature of the membrane as proposed by the classical fluid mosaic model. Experiments indicate that biomembranes of eukaryotic cells may be laterally organized into small nanoscopic domains, called rafts, which are rich in sphingomyelin and cholesterol. It is largely believed that this in-plane organization is essential for a variety of physiological functions such as signaling, recruitment of specific proteins and endocytosis. However, elucidation of the fundamental issues including the mechanisms leading to the formation of lipid rafts, their stability, and their size remain difficult. This has reiterated the importance of understanding the equilibrium phase behavior and the kinetics of fluid multicomponent lipid membranes before attempts are made to find the effects of more complex mechanisms that may be involved in the formation and stability of lipid rafts. Current increase in interest in the domain formation in multicomponent membranes also stems from the experiments demonstrating fluid-fluid coexistence in mixtures of lipids and cholesterol and the success of several computational models in predicting their behavior. Here we review time dependent Ginzburg Landau model, dynamical triangulation Monte Carlo, and dissipative particle dynamics which are some of the methods that are commonly employed.

  6. Isentropic Compression of Multicomponent Mixtures of Fuels and Inert Gases

    NASA Technical Reports Server (NTRS)

    Barragan, Michelle; Julien, Howard L.; Woods, Stephen S.; Wilson, D. Bruce; Saulsberry, Regor L.

    2000-01-01

    In selected aerospace applications of the fuels hydrazine and monomethythydrazine, there occur conditions which can result in the isentropic compression of a multicomponent mixture of fuel and inert gas. One such example is when a driver gas such as helium comes out of solution and mixes with the fuel vapor, which is being compressed. A second example is when product gas from an energetic device mixes with the fuel vapor which is being compressed. Thermodynamic analysis has shown that under isentropic compression, the fuels hydrazine and monomethylhydrazine must be treated as real fluids using appropriate equations of state. The appropriate equations of state are the Peng-Robinson equation of state for hydrazine and the Redlich-Kwong-Soave equation of state for monomethylhydrazine. The addition of an inert gas of variable quantity and input temperature and pressure to the fuel compounds the problem for safety design or analysis. This work provides the appropriate thermodynamic analysis of isentropic compression of the two examples cited. In addition to an entropy balance describing the change of state, an enthalpy balance is required. The presence of multicomponents in the system requires that appropriate mixing rules are identified and applied to the analysis. This analysis is not currently available.

  7. Multicomponent, Rare-Earth-Doped Thermal-Barrier Coatings

    NASA Technical Reports Server (NTRS)

    Miller, Robert A.; Zhu, Dongming

    2005-01-01

    Multicomponent, rare-earth-doped, perovskite-type thermal-barrier coating materials have been developed in an effort to obtain lower thermal conductivity, greater phase stability, and greater high-temperature capability, relative to those of the prior thermal-barrier coating material of choice, which is yttria-partially stabilized zirconia. As used here, "thermal-barrier coatings" (TBCs) denotes thin ceramic layers used to insulate air-cooled metallic components of heat engines (e.g., gas turbines) from hot gases. These layers are generally fabricated by plasma spraying or physical vapor deposition of the TBC materials onto the metal components. A TBC as deposited has some porosity, which is desirable in that it reduces the thermal conductivity below the intrinsic thermal conductivity of the fully dense form of the material. Undesirably, the thermal conductivity gradually increases because the porosity gradually decreases as a consequence of sintering during high-temperature service. Because of these and other considerations such as phase transformations, the maximum allowable service temperature for yttria-partially stabilized zirconia TBCs lies in the range of about 1,200 to 1,300 C. In contrast, the present multicomponent, rare-earth-doped, perovskite-type TBCs can withstand higher temperatures.

  8. Twisted Landau damping rates in multi-component dusty plasmas

    NASA Astrophysics Data System (ADS)

    Ali, S.; Bukhari, S.; Mendonca, J. T.

    2016-03-01

    Keeping in view the kinetic treatment for plasma particles, the electrostatic twisted dust-acoustic (DA) and dust-ion-acoustic (DIA) waves are investigated in a collisionless unmagnetized multi-component dusty plasma, whose constituents are the electrons, singly ionized positive ions, and negatively charged massive dust particulates. With this background, the Vlasov-Poisson equations are coupled together to derive a generalized dielectric constant by utilizing the Laguerre-Gaussian perturbed distribution function and electrostatic potential in the paraxial limit. The dispersion and damping rates of twisted DA and DIA waves are analyzed with finite orbital angular momentum states in a multi-component dusty plasma. Significant modifications concerning the real wave frequencies and damping rates appeared with varying twisted dimensionless parameter and dust concentration. In particular, it is shown that dust concentration enhances the phase speed of the DIA waves in contrary to DA waves, whereas the impact of twisted parameter reduces the frequencies of both DA and DIA waves. The results should be useful for the understanding of particle transport and trapping phenomena caused by wave excitation in laboratory dusty plasmas.

  9. High Glass Transition Temperature Renewable Polymers via Biginelli Multicomponent Polymerization.

    PubMed

    Boukis, Andreas C; Llevot, Audrey; Meier, Michael A R

    2016-04-01

    A novel and straightforward one-pot multicomponent polycondensation method was established in this work. The Biginelli reaction is a versatile multicomponent reaction of an aldehyde, a β-ketoester (acetoacetate) and urea, which can all be obtained from renewable resources, yielding diversely substituted 3,4-dihydropyrimidin-2(1H)-ones (DHMPs). In this study, renewable diacetoacetate monomers with different spacer chain lengths (C3, C6, C10, C20) were prepared via simple transesterification of renewable diols and commercial acetoacetates. The diacetoacetate monomers were then reacted with renewable dialdehydes, i.e., terephthalaldehyde and divanillin in a Biginelli type step-growth polymerization. The obtained DHMP polymers (polyDHMPs) displayed high molar masses, high glass transition temperatures (Tg) up to 203 °C and good thermal stability (Td5%) of 280 °C. The Tg of the polyDHMPs could be tuned by variation of the structure of the dialdehyde or the diacetoacetate component. PMID:26800511

  10. Biased multicomponent reactions to develop novel bromodomain inhibitors.

    PubMed

    McKeown, Michael R; Shaw, Daniel L; Fu, Harry; Liu, Shuai; Xu, Xiang; Marineau, Jason J; Huang, Yibo; Zhang, Xiaofeng; Buckley, Dennis L; Kadam, Asha; Zhang, Zijuan; Blacklow, Stephen C; Qi, Jun; Zhang, Wei; Bradner, James E

    2014-11-13

    BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors. PMID:25314271

  11. Biased Multicomponent Reactions to Develop Novel Bromodomain Inhibitors

    PubMed Central

    2015-01-01

    BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors. PMID:25314271

  12. Smoking cessation after 12 months with multi-component therapy.

    PubMed

    Raich, Antònia; Martínez-Sánchez, Jose Maria; Marquilles, Emili; Rubio, Lídia; Fu, Marcela; Fernández, Esteve

    2015-01-01

    Smoking is one of the most important causes of morbidity and mortality in developed countries. One of the priorities of public health programmes is the reduction of its prevalence, which would involve millions of people quitting smoking, but cessation programs often have modest results, especially within certain population groups. The aim of this study was to analyze the variables determining the success of a multicomponent therapy programme for smoking cessation. We conducted the study in the Smoking Addiction Unit at the Hospital of Manresa, with 314 patients (91.4% of whom had medium or high-level dependency). We observed that higher educational level, not living with a smoker, following a multimodal programme or smoking cessation with psychological therapy, and pharmacological treatment are relevant factors for quitting smoking. Abstinence rates are not associated with other factors, such as sex, age, smoking behaviour characteristics or psychiatric history. The combination of pharmacological and psychological treatment increased success rates in multicomponent therapy. Psychological therapy only also obtained positive results, though somewhat more modest. PMID:25879476

  13. Microscopic characterization of individual particles from multicomponent ship exhaust.

    PubMed

    Popovicheva, Olga; Kireeva, Elena; Persiantseva, Natalia; Timofeev, Mikhail; Bladt, Henrike; Ivleva, Natalia P; Niessner, Reinhard; Moldanová, Jana

    2012-12-01

    Particles sampled from the main and auxiliary ship diesel engine exhausts during a measurement campaign aboard a cargo ship are studied by SEM and energy-dispersive X-ray (EDX) microanalysis. Cluster analysis (CA) is applied to characterize the particles by separating them into distinct groups of similar morphology and chemical composition, representative of the particle types in the exhaust from the main and auxiliary engines. Raman microspectroscopy, Fourier transform infrared (FTIR) spectroscopy, inductively coupled plasma mass spectrometry and ion chromatography provide the criteria for the clustering of a large data set of individual particles. To identify chemical and morphological features of heavy and distillate fuel oil-derived PM emissions, micromarkers discriminating between the different types of emitted particles are proposed. These micromarkers could enable the classification of multicomponent aerosols according to a source type. This characterization of complex multicomponent aerosols emitted by ship diesel engines improves the quantification of the contribution of shipping to ambient air particulates, and can help to identify a source type in apportionment studies. PMID:23090431

  14. Microreactor Technology as an Efficient Tool for Multicomponent Reactions

    NASA Astrophysics Data System (ADS)

    Cukalovic, Ana; Monbaliu, Jean-Christophe M. R.; Stevens, Christian V.

    Multicomponent reactions are an important tool in organic synthesis as they often allow the circumvention of multistep procedures by combining three or more molecules into one structure in a single step. An additional asset of the approach is the significant increase of the combinatorial possibilities, since a modification of the final product is easily accomplished by implementing minor changes in the reaction setup; this obviously allows considerable savings in time and resources. These advantages are of particular interest in pharmaceutical research for the construction of libraries. In order to increase the sustainability of chemical processes, the field is intensively explored, and novel reactions are frequently reported. Microreactor technology also offers a contemporary way of conducting chemical reactions in a more sustainable fashion due to the miniaturization and increased safety, and also in a technically improved manner due to intensified process efficiency. This relatively new technology is implemented in novel and improved applications and is getting more and more used in chemical research. The combination of the benefits from the two approaches clearly presents an attractive reaction design, and this chapter presents an overview of the reported examples in which the microreactor technology and the multicomponent approach are combined, usually with dramatically improved results compared to those previously reported.

  15. Criticality in multicomponent spherical models: Results and cautions

    NASA Astrophysics Data System (ADS)

    Aqua, Jean-Noël; Fisher, Michael E.

    2009-01-01

    To enable the study of criticality in multicomponent fluids, the standard spherical model is generalized to describe an S -species hard-core lattice gas. On introducing S spherical constraints, the free energy may be expressed generally in terms of an S×S matrix describing the species interactions. For binary systems, thermodynamic properties have simple expressions, while all the pair correlation functions are combinations of just two eigenmodes. When only hard-core and short-range overall attractive interactions are present, a choice of variables relates the behavior to that of one-component systems. Criticality occurs on a locus terminating a coexistence surface; however, except at some special points, an unexpected “demagnetization effect” suppresses the normal divergence of susceptibilities at criticality and distorts two-phase coexistence. This effect, unphysical for fluids, arises from a general lack of symmetry and from the vectorial and multicomponent character of the spherical model. Its origin can be understood via a mean-field treatment of an XY spin system below criticality.

  16. Criticality in multicomponent spherical models: results and cautions.

    PubMed

    Aqua, Jean-Noël; Fisher, Michael E

    2009-01-01

    To enable the study of criticality in multicomponent fluids, the standard spherical model is generalized to describe an S -species hard-core lattice gas. On introducing S spherical constraints, the free energy may be expressed generally in terms of an SxS matrix describing the species interactions. For binary systems, thermodynamic properties have simple expressions, while all the pair correlation functions are combinations of just two eigenmodes. When only hard-core and short-range overall attractive interactions are present, a choice of variables relates the behavior to that of one-component systems. Criticality occurs on a locus terminating a coexistence surface; however, except at some special points, an unexpected "demagnetization effect" suppresses the normal divergence of susceptibilities at criticality and distorts two-phase coexistence. This effect, unphysical for fluids, arises from a general lack of symmetry and from the vectorial and multicomponent character of the spherical model. Its origin can be understood via a mean-field treatment of an XY spin system below criticality. PMID:19257012

  17. Statistical physics of multicomponent alloys using KKR-CPA

    NASA Astrophysics Data System (ADS)

    Khan, Suffian N.; Staunton, J. B.; Stocks, G. M.

    2016-02-01

    We apply variational principles from statistical physics and the Landau theory of phase transitions to multicomponent alloys using the multiple-scattering theory of Korringa-Kohn-Rostoker (KKR) and the coherent potential approximation (CPA). This theory is a multicomponent generalization of the S(2 ) theory of binary alloys developed by G. M. Stocks, J. B. Staunton, D. D. Johnson, and others. It is highly relevant to the chemical phase stability of high-entropy alloys as it predicts the kind and size of finite-temperature chemical fluctuations. In doing so, it includes effects of rearranging charge and other electronics due to changing site occupancies. When chemical fluctuations grow without bound, an absolute instability occurs and a second-order order-disorder phase transition may be inferred. The S(2 ) theory is predicated on the fluctuation-dissipation theorem; thus we derive the linear response of the CPA medium to perturbations in site-dependent chemical potentials in great detail. The theory lends itself to a natural interpretation in terms of competing effects: entropy driving disorder and favorable pair interactions driving atomic ordering. To further clarify interpretation, we present results for representative ternary alloys CuAgAu, NiPdPt, RhPdAg, and CoNiCu within a frozen charge (or band-only) approximation. These results include the so-called Onsager mean-field correction that extends the temperature range for which the theory is valid.

  18. Convective stability of multicomponent fluids in the thermogravitational column.

    PubMed

    Ryzhkov, Ilya I; Shevtsova, Valentina M

    2009-02-01

    A comprehensive linear stability analysis of convection in the thermogravitational column is first performed for multicomponent fluids. Two types of perturbations are investigated: Longitudinal waves propagating in vertical direction of the column and transversal waves propagating perpendicular to the vertical axis and temperature gradient. The stability problems are reduced to those without cross-diffusion effect by a special transformation. The calculations are performed for binary and ternary mixtures by the Galerkin method. It is found that in binary fluids, the onset of longitudinal instability can be monotonic or oscillatory depending on the separation ratio, which characterizes the Soret effect. The difference between stability characteristics of binary and ternary fluids is associated with different diffusion times of components in a ternary system. It is shown that the mechanism of transversal instability is related to the unstable density stratification in the column (in total or due to individual components). The unstable stratification can only be realized in fluids with negative Soret effect. The analogue of exchange of stabilities principle for a plane column with a multicomponent fluid is proved. The obtained results indicate that the thermogravitational column can be used for measuring diffusion and thermal diffusion coefficients in ternary and higher mixtures with one or several components having negative Soret effect. PMID:19391841

  19. Effective binary theory of multi-component nucleation

    SciTech Connect

    Kalikmanov, V. I.

    2015-03-28

    Classical theory of multi-component nucleation [O. Hirschfelder, J. Chem. Phys. 61, 2690 (1974)] belongs to the class of the so-called intractable problems: it requires computational time which is an exponential function of the number of components N. For a number of systems of practical interest with N > 10, the brute-force use of the classical theory becomes virtually impossible and one has to resort to an effective medium approach. We present an effective binary model which captures important physics of multi-component nucleation. The distinction between two effective species is based on the observation that while all N components contribute to the cluster thermodynamic properties, there is only a part of them which trigger the nucleation process. The proposed 2D-theory takes into account adsorption by means of the Gibbs dividing surface formalism and uses statistical mechanical considerations for the treatment of small clusters. Theoretical predictions for binary-, ternary-, and 14-component mixtures are compared with available experimental data and other models.

  20. Acute and subacute effects of miconazole nitrate on hepatic styrene oxide hydrolase and cytochrome P-450-dependent monooxygenase activities in male and female AKR/J mice.

    PubMed

    James, M O

    1988-08-01

    The imidazole-containing anti-fungal drug, miconazole nitrate, was shown to enhance hepatic microsomal styrene oxide hydrolase and inhibit several cytochrome P-450-dependent monooxygenase activities in the AKR/J mouse. Miconazole was a more potent inhibitor of cytochrome P-450-dependent monooxygenase activities in microsomes from male than female mice, and inhibitory potency also varied with substrate. When administered in vivo miconazole nitrate stimulated epoxide hydrolase activity, but had a substrate-dependent biphasic effect on cytochrome P-450-dependent monooxygenase activities. Monooxygenase activities with benzo[a]pyrene and benzphetamine were inhibited to varying degrees in liver homogenate and hepatic microsomes from mice sacrificed 45 min after miconazole administration. After repeated administration of miconazole, liver weight, microsomal protein yield and cytochrome P-450 were increased, as were specific monooxygenase activities with ethoxycoumarin and ethoxyresorufin, but benzphetamine N-demethylase activity was decreased. These results suggested that a metabolite of miconazole was responsible for the inhibition of benzphetamine N-demethylase. It was of special interest that ethoxyresorufin O-deethylase activity was induced in the AKR/J mouse by miconazole, since the AKR/J mouse is not responsive to induction by aromatic hydrocarbons. PMID:3394155

  1. Nonstoichiometric nucleation and growth of multicomponent nanocrystals in solution.

    PubMed

    Min, Yuho; Kwak, Junghyeok; Soon, Aloysius; Jeong, Unyong

    2014-10-21

    The ability to assemble nanoscale functional building blocks is a useful and modular way for scientists to design valuable materials with specific physical and chemical properties. Chemists expect multicomponent, heterostructured nanocrystals to show unique electrical, thermal, and optical properties not seen in homogeneous, single-phase nanocrystals. Although researchers have made remarkable advances in heterogeneous nucleation and growth, design of synthetic conditions for obtaining nanocrystals with a target composition and shape is still a big challenge. There are several outstanding issues that chemists need to address before they can successfully carry out the design-based synthesis of multicomponent nanocrystals. For instance, small changes in the reaction parameters, such as the precursor, solvent, surfactant, reducing agent, and the reaction temperature, often result in changes in the structure and chemical composition of the final product. Although scientists do not fully understand the mechanisms underlying the nucleation and growth processes involved in the synthesis of these multicomponent nanocrystals, recent progress in understanding of the thermodynamic and kinetic factors have improved our control over their final structure and chemical composition. In this Account, we summarize our recent advances in understanding of the nucleation and growth mechanisms involved in the solution-based synthesis of multicomponent nanocrystals. We also discuss the various challenges encountered in their synthesis, emphasizing what still needs special consideration. We first discuss the three different nucleation paths from a thermodynamics perspective: amorphous nucleation, crystalline nucleation, and two-step nucleation. Amorphous nucleation and two-step nucleation involve the generation of nonstoichiometric nuclei. We initiate this process mainly by introducing an imbalance in the concentrations of the reduced elements. When the nonstoichiometric nuclei grow, we

  2. Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases

    PubMed Central

    Forsberg, Zarah; Mackenzie, Alasdair K.; Sørlie, Morten; Røhr, Åsmund K.; Helland, Ronny; Arvai, Andrew S.; Vaaje-Kolstad, Gustav; Eijsink, Vincent G. H.

    2014-01-01

    For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu2+ (12–31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms. PMID:24912171

  3. Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation

    PubMed Central

    Arfi, Yonathan; Shamshoum, Melina; Rogachev, Ilana; Peleg, Yoav; Bayer, Edward A.

    2014-01-01

    Efficient conversion of cellulose into soluble sugars is a key technological bottleneck limiting efficient production of plant-derived biofuels and chemicals. In nature, the process is achieved by the action of a wide range of cellulases and associated enzymes. In aerobic microrganisms, cellulases are secreted as free enzymes. Alternatively, in certain anaerobic microbes, cellulases are assembled into large multienzymes complexes, termed “cellulosomes,” which allow for efficient hydrolysis of cellulose. Recently, it has been shown that enzymes classified as lytic polysaccharide monooxygenases (LPMOs) were able to strongly enhance the activity of cellulases. However, LPMOs are exclusively found in aerobic organisms and, thus, cannot benefit from the advantages offered by the cellulosomal system. In this study, we designed several dockerin-fused LPMOs based on enzymes from the bacterium Thermobifida fusca. The resulting chimeras exhibited activity levels on microcrystalline cellulose similar to that of the wild-type enzymes. The dockerin moieties of the chimeras were demonstrated to be functional and to specifically bind to their corresponding cohesin partner. The chimeric LPMOs were able to self-assemble in designer cellulosomes alongside an endo- and an exo-cellulase also converted to the cellulosomal mode. The resulting complexes showed a 1.7-fold increase in the release of soluble sugars from cellulose, compared with the free enzymes, and a 2.6-fold enhancement compared with free cellulases without LPMO enhancement. These results highlight the feasibility of the conversion of LPMOs to the cellulosomal mode, and that these enzymes can benefit from the proximity effects generated by the cellulosome architecture. PMID:24927597

  4. Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases.

    PubMed

    Westereng, Bjørge; Agger, Jane Wittrup; Horn, Svein J; Vaaje-Kolstad, Gustav; Aachmann, Finn L; Stenstrøm, Yngve H; Eijsink, Vincent G H

    2013-01-01

    We present an evaluation of HPLC-based analytical tools for the simultaneous analysis of native and oxidized cello-oligosaccharides, which are products of enzymatic cellulose degradation. Whereas cello-oligosaccharides arise from cellulose depolymerization by glycoside hydrolases, oxidized cello-oligosaccharides are produced by cellobiose dehydrogenase and the recently identified copper dependent lytic polysaccharide monooxygenases (LPMOs) currently classified as CBM33 and GH61. The latter enzymes are wide-spread and expected to play crucial roles in further development of efficient enzyme technology for biomass conversion. Three HPLC approaches with well documented performance in the field of oligosaccharide analysis have been investigated: high-performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and porous graphitized carbon liquid chromatography (PGC-LC). HPAEC with pulsed amperometric detection (PAD) was superior for analysis of oxidized oligosaccharides, combining the best separation with superior sensitivity for oligosaccharide species with a degree of polymerization (DP) ranging from 1 to 10. Furthermore, the HPAEC method can be optimized for operation in a high-throughput manner (run time 10 min). Both PGC-LC and HILIC allow reasonable run times (41 and 25 min, respectively), with acceptable separation, but suffer from poor sensitivity compared to HPAEC-PAD. On the other hand, PGC-LC and HILIC benefit from being fully compatible with online mass spectrometry. Using an LC-MS setup, these methods will deliver much better sensitivity than what can be obtained with conventional detectors such as ultraviolet-, charged aerosol-, or evaporative light scattering and may reach sensitivities similar to or even better than what is obtained in HPAEC-PAD. Pure oxidized cello-oligosaccharide standards, ranging from DP2 to DP5, were obtained by semi-preparative PGC and characterized by MS and NMR analysis. PMID:23246088

  5. Substrate Hydroxylation in Methane Monooxygenase: Quantitative Modeling via Mixed Quantum Mechanics/ Molecular Mechanics Techniques

    SciTech Connect

    Gherman, Benjamin F.; Lippard, Stephen J.; Friesner, Richard A.

    2005-01-26

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Using broken-symmetry unrestricted density functional theory quantum mechanical (QM) methods in concert with mixed quantum mechanics/molecular mechanics (QM/MM) methods, the hydroxylation of methane and substituted methanes by intermediate Q in methane monooxygenase hydroxylase (MMOH) has been quantitatively modeled. This protocol allows the protein environment to be included throughout the calculations and its effects (electrostatic, van der Waals, strain) upon the reaction to be accurately evaluated. With the current results, recent kinetic data for CH₃X (X ) H, CH₃, OH, CN, NO₂) substrate hydroxylation in MMOH (Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 8770-8771) can be rationalized. Results for methane, which provide a quantitative test of the protocol, including a substantial kinetic isotope effect (KIE), are in reasonable agreement with experiment. Specific features of the interaction of each of the substrates with MMO are illuminated by the QM/MM modeling, and the resulting effects upon substrate binding are quantitatively incorporated into the calculations. The results as a whole point to the success of the QM/MM methodology and enhance our understanding of MMOH catalytic chemistry. We also identify systematic errors in the evaluation of the free energy of binding of the Michaelis complexes of the substrates, which most likely arise from inadequate sampling and/or the use of harmonic approximations to evaluate the entropy of the complex. More sophisticated sampling methods will be required to achieve greater accuracy in this aspect of the calculation.

  6. The Catalytic Copper of Peptidylglycine alpha-Hydroxylating Monooxygenase also Plays a Critical Structural Role

    SciTech Connect

    Siebert,X.; Eipper, B.; Mains, R.; Prigge, S.; Blackburn, N.; Amzel, L.

    2005-01-01

    Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-Hydroxylating Monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met314 and two His residues as ligands. Mutation of Met314 to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met314 as a CuM ligand, we determined the structure of the M314I-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The M314I-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the M314I-PHM crystal form and showed that it does not differ from the structure of wt-PHM in the original crystals. M314I-PHM was also shown to be less stable than wt-PHM by Differential Scanning Calorimetry (DSC). Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.

  7. Purification and properties of 4-aminobenzoate hydroxylase, a new monooxygenase from Agaricus bisporus.

    PubMed

    Tsuji, H; Ogawa, T; Bando, N; Sasaoka, K

    1986-10-01

    A new FAD-dependent monooxygenase, 4-aminobenzoate hydroxylase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate and forms 4-hydroxyaniline in the presence of NAD(P)H and O2 has been purified to homogeneity by ammonium sulfate fractionation, affinity chromatography, chromatofocusing, and Sephadex G-100 chromatography from Agaricus bisporus, a common edible mushroom. The molecular weight of the enzyme, which consists of a single polypeptide, is 49,000. The enzyme contains 0.91 mol of FAD/mol of enzyme. Stoichiometric studies show that 1 mol of 4-aminobenzoate is converted to an equimolecular amount of 4-hydroxyaniline and CO2 with the consumption of 1 mol each of NADH and molecular oxygen. Results obtained isotopically with 18O2 show that one atom of molecular oxygen is incorporated into 4-hydroxyaniline formed from 4-aminobenzoate. The enzyme is most active between pH 6.5 and 8.0 in the oxidation of NADH and between pH 6.0 and 7.5 in the case of NADPH. The Km values for 4-aminobenzoate, NADH, and O2 are 20.4, 13.6, and 200 microM, respectively, and that for NADPH is 133 microM. Other substituted benzoates with free amino and carboxyl groups in the ortho or para position (e.g. 4-aminosalicylate and anthranilate) serve as substrates for hydroxylation, but, in these cases, H2O2 is formed simultaneously with the hydroxylation. The enzyme is insensitive to the chelators of iron and copper, sodium arsenite, and KCN. Heavy metal ions and p-chloromercuribenzoate severely inhibit the enzyme enzyme PMID:3489713

  8. Pharmacological kynurenine 3-monooxygenase enzyme inhibition significantly reduces neuropathic pain in a rat model.

    PubMed

    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. PMID:26524415

  9. Evaluating cytochrome P450 in birds by monooxygenases and immunohistochemistry: possible nonlethal assessment by skin immunohistochemistry

    USGS Publications Warehouse

    Melancon, M.J.; Kutay, A.L.; Woodin, Bruce R.; Stegeman, John J.

    2000-01-01

    Six month old Lesser Scaup and nestling Tree Swallows were injected intraperitoneally with beta-naphthoflavone (BNF) or vehicle. Nestling Tree Swallows were also collected from five sites with differing levels of contaminants. Liver samples were taken and stored at -80C until microsome preparation and monooxygenase (MO) assay. Skin and heart samples were placed in buffered formalin until immunohistochemical (IMHC) analysis for cytochrome P4501A (CYP1A). Scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 20- to 65-fold increases in four MOs. Responses of two of the four MOs were as high at 20 mg/kg as at 100mg/kg. There was no IMHC response in the vehicle-injected ducks, while in skin the IMHC response was the same for both dose levels of BNF and in heart there was response in two of four samples at 20 mg/kg and in all five samples at 100mg/kg. Tree Swallows injected with BNF at 100, but not at 20 mg/kg showed significant increases (ca.5-fold) in two MO activities. There was no IMHC response in control swallows. In skin and heart there were IMHC responses in one of five swallows at 20 mg/kg and four of five swallows at 100mg/kg. There was poor correlation between individual skin IMHC responses and MO activities and PCB concentrations in 47 field-collected Tree Swallow samples, but 14 of the 16 skin samples with positive IMHC responses were from the location with the highest MO activities and PCB concentrations. Although present data do not allow construction of significant dose response curves, the responses in skin make it well worth continuing study on this potential nonlethal technique for biomonitoring contaminant exposure of birds.

  10. Ascorbate depletion as a consequence of product recycling during dopamine. beta. -monooxygenase catalyzed selenoxidation

    SciTech Connect

    May, S.W.; Herman, H.H.; Roberts, S.F.; Ciccarello, M.C.

    1987-03-24

    The competence of dopamine ..beta..-monooxygenase (DBM) to process selenide substrates was investigated, in anticipation that the expected selenoxide products would exhibit unique reactivity and redox properties. The prototypical selenide phenyl 2-aminoethyl selenide (PAESe) was synthesized and shown to be a substrate for DBM with the characteristic e/O/sub 2/ ratio of 2:1 for monooxygenation. The kinetic parameters for oxygenation of PAESe were found to be similar to those for the DBM-catalyzed sulfoxidation of the cognate sulfide phenyl 2-aminoethyl sulfide, and selenoxidation was stimulated by fumarate in a manner similar to other well-characterized DBM monooxygenation reactions. Identification of phenyl 2-aminoethyl selenoxide (PAESeO) as the enzymatic product was accomplished by the demonstration of coincident elution of authentic PAESeO with the enzymatic product in three significantly different HPLC systems. PAESeO was found to oxidize ascorbic acid with the concomitant and stoichiometric reduction of PAESeO back to the selenide, PAESe. As a consequence of this nonenzymatic reaction, ascorbate-supported DBM turnover was prematurely terminated under standard assay conditions due to depletion of reduced ascorbate. The kinetics of the redox reaction between PAESeO and ascorbate were investigated with a spectrophotometric assay of ascorbate at 300 nm, and a second-order rate constant of 3.4 M/sup -1/ s/sup -1/ was determined at pH 5.0, 25/sup 0/C. Spectrophotometric assay of cytochrome c (cyt c) reduction at 550 nm during the oxidation of ascorbate by PAESeO demonstrated that no cyt c trappable semidehydroascorbate was produced in this nonenzymatic reaction.

  11. Fungal Cytochrome P450 Monooxygenases: Their Distribution, Structure, Functions, Family Expansion, and Evolutionary Origin

    PubMed Central

    Chen, Wanping; Lee, Mi-Kyung; Jefcoate, Colin; Kim, Sun-Chang; Chen, Fusheng; Yu, Jae-Hyuk

    2014-01-01

    Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi. PMID:24966179

  12. Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones

    SciTech Connect

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Krueger, Sharon K.; Stevens, J. Fred; Kedzie, Karen; Fang, Wenkui K.; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E.

    2014-07-15

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC–MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with K{sub m}s ranging from 7 to 160 μM and turnover numbers of 30–40 min{sup −1}. The product formed was identified by LC–MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.

  13. Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases.

    PubMed

    Forsberg, Zarah; Mackenzie, Alasdair K; Sørlie, Morten; Røhr, Åsmund K; Helland, Ronny; Arvai, Andrew S; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

    2014-06-10

    For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu(2+) (12-31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms. PMID:24912171

  14. The Nature of the Reaction Intermediates in the FAD-Dependent Epoxidation Mechanism of Styrene Monooxygenase*

    PubMed Central

    Kantz, Auric; Gassner, George T.

    2010-01-01

    Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of an NADH-specific flavin reductase (SMOB) and FAD-specific styrene epoxidase (NSMOA). NSMOA binds tightly to reduced FAD and catalyzes the stereospecific addition of one atom of molecular oxygen to the vinyl side chain of styrene in the enantioselective synthesis of S-styrene oxide. In this mechanism, molecular oxygen first reacts with NSMOA(FADred) to yield an FAD C(4a)- peroxide intermediate. This species is non-fluorescent and has an absorbance maximum of 382 nm. Styrene then reacts with the peroxide intermediate with a second order rate constant of 2.6 × 106 ± 0.1 × 106 M−1 s−1 to yield a fluorescent intermediate with an absorbance maximum of 368 nm. We compute an activation free energy of 8.7 kcal.mol∙−1 for the oxygenation step in good agreement with that expected for a peroxide-catalyzed epoxidation, and acid-quenched samples recovered at defined time points in the single-turnover reaction indicate that styrene oxide synthesis is coincident with the formation phase of the fluorescent intermediate. These findings support FAD C(4a)-peroxide as the oxygen atom donor and identity of the fluorescent intermediate as an FAD C(4a)-hydroxide product of the styrene epoxidation. Overall, four pH-dependent rate constants corresponding to peroxyflavin formation (pKa = 7.2), styrene epoxidation (pKa = 7.7), styrene oxide dissociation (pKa = 8.3), and hydroxyflavin dehydration (pKa 7.6) are needed to fit the single-turnover kinetics. PMID:21166448

  15. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates

    PubMed Central

    Kong, Wenwen; Li, Jing; Yu, Qingyue; Cang, Wei; Xu, Rui; Wang, Yang; Ji, Wei

    2016-01-01

    Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates—S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates—was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes. PMID:27621741

  16. Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Blazyk, Jessica L; Lippard, Stephen J

    2004-02-13

    Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the conversion of methane to methanol. A reductase (MMOR), which contains [2Fe-2S] and FAD cofactors, facilitates electron transfer from NADH to the hydroxylase diiron active sites where dioxygen activation and substrate hydroxylation take place. By separately expressing the ferredoxin (MMORFd, MMOR residues 1-98) and FAD/NADH (MMOR-FAD, MMOR residues 99-348) domains of the reductase, nearly all biochemical properties of full-length MMOR are retained, except for interdomain electron transfer rates. To investigate the extent to which rapid electron transfer between domains might be restored and further to explore the modularity of MMOR, MMOR-Fd and MMOR-FAD were connected in a non-native fashion. Four different linker sequences were employed to create MMOR reversed-domain (MMOR-RD) constructs, MMOR(99-342)-linker-MMOR(2-98), with a domain connectivity observed in other homologous oxidoreductases. The optical, redox, and electron transfer properties of the four MMOR-RD proteins were characterized and compared with those of wild-type MMOR. The linker sequence plays a key role in controlling solvent accessibility to the FAD cofactor, as evidenced by perturbed flavin optical spectra, decreased FADox/FADsq redox potentials, and increased steady-state oxidase activities in three of the constructs. Stopped-flow optical spectroscopy revealed slow interdomain electron transfer (k < 0.04 s(-1) at 4 degrees C, compared with 90 s(-1) for wild-type MMOR) for all three MMOR-RD proteins with 7-residue linkers. A long (14-residue), flexible linker afforded much faster electron transfer between the FAD and [2Fe-2S] cofactors (k = 0.9 s(-1) at 4 degrees C). PMID:14613937

  17. Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath).

    PubMed

    Gassner, G T; Lippard, S J

    1999-09-28

    The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase. PMID:10504247

  18. Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Kopp, D A; Gassner, G T; Blazyk, J L; Lippard, S J

    2001-12-11

    Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system. PMID:11732913

  19. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase.

    PubMed

    Paszczynski, Andrzej J; Paidisetti, Ravindra; Johnson, Andrew K; Crawford, Ronald L; Colwell, Frederick S; Green, Tonia; Delwiche, Mark; Lee, Hope; Newby, Deborah; Brodie, Eoin L; Conrad, Mark

    2011-11-01

    The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN. PMID:21360114

  20. Temperature-sensitive albino gene TCD5, encoding a monooxygenase, affects chloroplast development at low temperatures.

    PubMed

    Wang, Yufeng; Zhang, Jianhui; Shi, Xiaoliang; Peng, Yu; Li, Ping; Lin, Dongzhi; Dong, Yanjun; Teng, Sheng

    2016-09-01

    Chloroplasts are essential for photosynthesis and play critical roles in plant development. In this study, we characterized the temperature-sensitive chlorophyll-deficient rice mutant tcd5, which develops albino leaves at low temperatures (20 °C) and normal green leaves at high temperatures (32 °C). The development of chloroplasts and etioplasts is impaired in tcd5 plants at 20 °C, and the temperature-sensitive period for the albino phenotype is the P4 stage of leaf development. The development of thylakoid membranes is arrested at the mid-P4 stage in tcd5 plants at 20 °C. We performed positional cloning of TCD5 and then complementation and knock-down experiments, and the results showed that the transcript LOC_Os05g34040.1 from the LOC_Os05g34040 gene corresponded to the tcd5 phenotype. TCD5 encodes a conserved plastid-targeted monooxygenase family protein which has not been previously reported associated with a temperature-sensitive albino phenotype in plants. TCD5 is abundantly expressed in young leaves and immature spikes, and low temperatures increased this expression. The transcription of some genes involved in plastid transcription/translation and photosynthesis varied in the tcd5 mutant. Although the phenotype and temperature dependence of the TCD5 orthologous mutant phenotype were different in rice and Arabidopsis, OsTCD5 could rescue the phenotype of the Arabidopsis mutant, suggesting that TCD5 function is conserved between monocots and dicots. PMID:27531886

  1. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

    PubMed

    Bennett, Kristen; Sadler, Natalie C; Wright, Aaron T; Yeager, Chris; Hyman, Michael R

    2016-04-01

    Nitrosomonas europaea is 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 O2 uptake by N. europaea by 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 matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

  2. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates.

    PubMed

    Kong, Wenwen; Li, Jing; Yu, Qingyue; Cang, Wei; Xu, Rui; Wang, Yang; Ji, Wei

    2016-01-01

    Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates-S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates-was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes. PMID:27621741

  3. Temperature-sensitive albino gene TCD5, encoding a monooxygenase, affects chloroplast development at low temperatures

    PubMed Central

    Wang, Yufeng; Zhang, Jianhui; Shi, Xiaoliang; Peng, Yu; Li, Ping; Lin, Dongzhi; Dong, Yanjun; Teng, Sheng

    2016-01-01

    Chloroplasts are essential for photosynthesis and play critical roles in plant development. In this study, we characterized the temperature-sensitive chlorophyll-deficient rice mutant tcd5, which develops albino leaves at low temperatures (20 °C) and normal green leaves at high temperatures (32 °C). The development of chloroplasts and etioplasts is impaired in tcd5 plants at 20 °C, and the temperature-sensitive period for the albino phenotype is the P4 stage of leaf development. The development of thylakoid membranes is arrested at the mid-P4 stage in tcd5 plants at 20 °C. We performed positional cloning of TCD5 and then complementation and knock-down experiments, and the results showed that the transcript LOC_Os05g34040.1 from the LOC_Os05g34040 gene corresponded to the tcd5 phenotype. TCD5 encodes a conserved plastid-targeted monooxygenase family protein which has not been previously reported associated with a temperature-sensitive albino phenotype in plants. TCD5 is abundantly expressed in young leaves and immature spikes, and low temperatures increased this expression. The transcription of some genes involved in plastid transcription/translation and photosynthesis varied in the tcd5 mutant. Although the phenotype and temperature dependence of the TCD5 orthologous mutant phenotype were different in rice and Arabidopsis, OsTCD5 could rescue the phenotype of the Arabidopsis mutant, suggesting that TCD5 function is conserved between monocots and dicots. PMID:27531886

  4. The Metal Centers of Particulate Methane Monooxygenase from Methylosinus trichosporium OB3b

    SciTech Connect

    Hakemian,A.; Kondapalli, K.; Telser, J.; Hoffman, B.; Stemmler, T.; Rosenzweig, A.

    2008-01-01

    Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 Angstroms. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

  5. Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

    PubMed

    Pham, Minh D; Lin, Ya-Ping; Van Vuong, Quan; Nagababu, Penumaka; Chang, Brian T-A; Ng, Kok Yaoh; Chen, Chein-Hung; Han, Chau-Chung; Chen, Chung-Hsuan; Li, Mai Suan; Yu, Steve S-F; Chan, Sunney I

    2015-12-01

    Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO. PMID:26275807

  6. The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b.

    PubMed

    Hakemian, Amanda S; Kondapalli, Kalyan C; Telser, Joshua; Hoffman, Brian M; Stemmler, Timothy L; Rosenzweig, Amy C

    2008-07-01

    Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers. PMID:18540635

  7. Transcriptomic profiling of Methylococcus capsulatus (Bath) during growth with two different methane monooxygenases.

    PubMed

    Larsen, Øivind; Karlsen, Odd A

    2016-04-01

    Methylococcus capsulatus (Bath) is a methanotroph that possesses both a membrane-embedded (pMMO) and a soluble methane monooxygenase (sMMO). The expression of these two MMO's is tightly controlled by the availability of copper in the growth medium, but the underlying mechanisms and the number of genes involved in this switch in methane oxidation is not yet fully elucidated. Microarray analyses were used to assess the transcriptome in cells producing either pMMO or sMMO. A total of 137 genes were differentially expressed, with 87 genes showing a significant up-regulation during sMMO production. The majority of the differentially expressed genes could be assigned to functional roles in the energy metabolism and transport. Furthermore, three copper responding gene clusters were discovered, including an extended cluster that also harbors the genes for sMMO. Our data also indicates that major changes takes place in the respiratory chain between pMMO- and sMMO-producing cells, and that quinone are predominantly used as the electron donors for methane oxidation by pMMO. Intriguingly, a large proportion of the differentially expressed genes between pMMO- and sMMO-producing cells encode c-type cytochromes. By combining microarray- and mass spectrometry data, a total of 35 c-type cytochromes are apparently expressed in M. capsulatus when grown in nitrate mineral salt medium with methane as sole energy and carbon source, and the expression of 21 of these respond to the availability of copper. Interestingly, several of these c-type cytochromes are recovered from the cell surface, suggesting that extracellular electron transfers may occur in M. capsulatus. PMID:26687591

  8. Flavin-Containing Monooxygenase S-Oxygenation of a Series of Thioureas and Thiones

    PubMed Central

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Krueger, Sharon K.; Stevens, J. Fred; Kedzie, Karen; Fang, Ken; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E.

    2014-01-01

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC-MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with Kms ranging from 7–160 μM and turnover numbers of 30–40 min−1. The product formed was identified by LC-MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1. PMID:24727368

  9. Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones.

    PubMed

    Henderson, Marilyn C; Siddens, Lisbeth K; Krueger, Sharon K; Stevens, J Fred; Kedzie, Karen; Fang, Wenkui K; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E

    2014-07-15

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC-MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2-7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with Kms ranging from 7 to 160 μM and turnover numbers of 30-40 min(-1). The product formed was identified by LC-MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1. PMID:24727368

  10. Mammalian flavin-containing monooxygenase (FMO) as a source of hydrogen peroxide.

    PubMed

    Siddens, Lisbeth K; Krueger, Sharon K; Henderson, Marilyn C; Williams, David E

    2014-05-01

    Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O₂ consumed as H₂O₂ upon addition of NADPH. Addition of substrate had little effect on H₂O₂ production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H₂O₂. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H₂O₂, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H₂O₂ generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H₂O₂ release. Catalase did not reduce levels of H₂O₂ with either FMO2.1 or FMO3 but inhibited H₂O₂ generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H₂O₂. PMID:24561181

  11. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

    SciTech Connect

    Andrzej J. Paszczynski; Ravindra Paidisetti; Andrew K. Johnson; Ronald L. Crawford; Frederick S. Colwell; Tonia Green; Mark Delwiche; Hope Lee; Deborah Newby; Eoin L. Brodie; Mark Conrad

    2011-11-01

    The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultraperformance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

  12. Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation.

    PubMed

    Arfi, Yonathan; Shamshoum, Melina; Rogachev, Ilana; Peleg, Yoav; Bayer, Edward A

    2014-06-24

    Efficient conversion of cellulose into soluble sugars is a key technological bottleneck limiting efficient production of plant-derived biofuels and chemicals. In nature, the process is achieved by the action of a wide range of cellulases and associated enzymes. In aerobic microrganisms, cellulases are secreted as free enzymes. Alternatively, in certain anaerobic microbes, cellulases are assembled into large multienzymes complexes, termed "cellulosomes," which allow for efficient hydrolysis of cellulose. Recently, it has been shown that enzymes classified as lytic polysaccharide monooxygenases (LPMOs) were able to strongly enhance the activity of cellulases. However, LPMOs are exclusively found in aerobic organisms and, thus, cannot benefit from the advantages offered by the cellulosomal system. In this study, we designed several dockerin-fused LPMOs based on enzymes from the bacterium Thermobifida fusca. The resulting chimeras exhibited activity levels on microcrystalline cellulose similar to that of the wild-type enzymes. The dockerin moieties of the chimeras were demonstrated to be functional and to specifically bind to their corresponding cohesin partner. The chimeric LPMOs were able to self-assemble in designer cellulosomes alongside an endo- and an exo-cellulase also converted to the cellulosomal mode. The resulting complexes showed a 1.7-fold increase in the release of soluble sugars from cellulose, compared with the free enzymes, and a 2.6-fold enhancement compared with free cellulases without LPMO enhancement. These results highlight the feasibility of the conversion of LPMOs to the cellulosomal mode, and that these enzymes can benefit from the proximity effects generated by the cellulosome architecture. PMID:24927597

  13. A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting α- and β-chitin.

    PubMed

    Nakagawa, Yuko S; Kudo, Madoka; Loose, Jennifer S M; Ishikawa, Takahiro; Totani, Kazuhide; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

    2015-03-01

    The lytic polysaccharide monooxygenases (LPMOs) have received considerable attention subsequent to their discovery because of their ability to boost the enzymatic conversion of recalcitrant polysaccharides. In the present study, we describe the enzymatic properties of SgLPMO10F, a small (15 kDa) auxilliary activity (AA) family 10 LPMO from Streptomyces griseus belonging to a clade of the phylogenetic tree without any characterized representative. The protein was expressed using a Brevibacillus-based expression system that had not been used previously for LPMO expression and that also ensures correct processing of the N-terminus crucial for LPMO activity. The enzyme was active towards both α- and β-chitin and showed stronger binding and a greater release of soluble oxidized products for the latter allomorph. In chitinase synergy assays, however, SgLPMO10F worked slightly better for α-chitin, increasing chitin solubilization yields by up to 30-fold and 20-fold for α- and β-chitin, respectively. Synergy experiments with various chitinases showed that the addition of SgLPMO10F leads to a substantial increase in the (GlcNAc)2 :GlcNAc product ratio, in reactions with α-chitin only. This underpins the structural differences between the substrates and also shows that, on α-chitin, SgLPMO10F affects the binding mode and/or degree of processivity of the chitinases tested. Variation in the only exposed aromatic residue in the substrate-binding surface of LPMO10s has previously been linked to preferential binding for α-chitin (exposed Trp) or β-chitin (exposed Tyr). Mutation of this residue, Tyr56, in SgLPMO10F to Trp had no detectable effect on substrate-binding preferences but, in synergy experiments, the mutant appeared to be more efficient on α-chitin. PMID:25605134

  14. Pseudomonas aeruginosa Population Structure Revisited

    PubMed Central

    Pirnay, Jean-Paul; Bilocq, Florence; Pot, Bruno; Cornelis, Pierre; Zizi, Martin; Van Eldere, Johan; Deschaght, Pieter; Vaneechoutte, Mario; Jennes, Serge; Pitt, Tyrone; De Vos, Daniel

    2009-01-01

    At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P

  15. Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a FAD-dependent monooxygenase for catalysis.

    PubMed

    Hollmann, Frank; Lin, Po-Chi; Witholt, Bernard; Schmid, Andreas

    2003-07-01

    Catalysis for chemical synthesis by cell-free monooxygenases necessitates an efficient and robust in situ regeneration system to supply the enzyme with reducing equivalents. We report on a novel approach to directly regenerate flavin-dependent monooxygenases. The organometallic complex [CpRh(bpy)(H(2)O)](2+) catalyzes the transhydrogenation reaction between formate and isoalloxazine-based cofactors such as FAD and FMN. Coupling this FADH(2) regeneration reaction to the FADH(2)-dependent styrene monooxygenase (StyA) resulted in a chemoenzymatic epoxidation reaction where the organometallic compound substitutes for the native reductase (StyB), the nicotinamide coenzyme (NAD), and an artificial NADH regeneration system such as formate dehydrogenase. Various styrene derivatives were converted into the essentially optically pure (S)-epoxides (ee > 98%). In addition, StyA was shown to be capable of performing sulfoxidation reactions. The productivity of the chemoenzymatic epoxidation reaction using 6.5 microM StyA reached up to 6.4 mM/h, corresponding to approximately 70% of a comparable fully enzymatic reaction using StyB, NADH, and formate dehydrogenase for regeneration. The coupling efficiency of the nonenzymatic regeneration reaction to enzymatic epoxidation was examined in detail, leading to an optimized reaction setup with minimized quenching of the electron supply for the epoxidation reaction. Thus, up to 60% of the reducing equivalents provided via [CpRh(bpy)(H(2)O)](2+) could be channeled into epoxide rather than hydrogen peroxide formation, allowing selective synthesis with high yields. PMID:12837091

  16. Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

    PubMed Central

    Kalidass, Bhagyalakshmi; Ul-Haque, Muhammad Farhan; Baral, Bipin S.; DiSpirito, Alan A.

    2014-01-01

    It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin. PMID:25416758

  17. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  18. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1

    PubMed Central

    Dyson, Zoe A.; Seviour, Robert J.; Tucci, Joseph

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  19. Pseudomonas helleri sp. nov. and Pseudomonas weihenstephanensis sp. nov., isolated from raw cow's milk.

    PubMed

    von Neubeck, M; Huptas, C; Glück, C; Krewinkel, M; Stoeckel, M; Stressler, T; Fischer, L; Hinrichs, J; Scherer, S; Wenning, M

    2016-03-01

    Analysis of the microbiota of raw cow's milk and semi-finished milk products yielded seven isolates assigned to the genus Pseudomonas that formed two individual groups in a phylogenetic analysis based on partial rpoD and 16S rRNA gene sequences. The two groups could be differentiated from each other and also from their closest relatives as well as from the type species Pseudomonas aeruginosa by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. ANIb values within the groups were higher than 97.3 %, whereas similarity values to the closest relatives were 85 % or less. The major cellular lipids of strains WS4917T and WS4993T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q-9 in both strains, with small amounts of Q-8 in strain WS4917T. The DNA G+C contents of strains WS4917T and WS4993T were 58.08 and 57.30 mol%, respectively. Based on these data, strains WS4917T, WS4995 ( = DSM 29141 = LMG 28434), WS4999, WS5001 and WS5002 should be considered as representatives of a novel species of the genus Pseudomonas, for which the name Pseudomonas helleri sp. nov. is proposed. The type strain of Pseudomonas helleri is strain WS4917T ( = DSM 29165T = LMG 28433T). Strains WS4993T and WS4994 ( = DSM 29140 = LMG 28438) should be recognized as representing a second novel species of the genus Pseudomonas, for which the name Pseudomonas weihenstephanensis sp. nov. is proposed. The type strain of Pseudomonas weihenstephanensis is strain WS4993T ( = DSM 29166T = LMG 28437T). PMID:26675012

  20. A Computationally Efficient Multicomponent Equilibrium Solver for Aerosols (MESA)

    SciTech Connect

    Zaveri, Rahul A.; Easter, Richard C.; Peters, Len K.

    2005-12-23

    This paper describes the development and application of a new multicomponent equilibrium solver for aerosol-phase (MESA) to predict the complex solid-liquid partitioning in atmospheric particles containing H+, NH4+, Na+, Ca2+, SO4=, HSO4-, NO3-, and Cl- ions. The algorithm of MESA involves integrating the set of ordinary differential equations describing the transient precipitation and dissolution reactions for each salt until the system satisfies the equilibrium or mass convergence criteria. Arbitrary values are chosen for the dissolution and precipitation rate constants such that their ratio is equal to the equilibrium constant. Numerically, this approach is equivalent to iterating all the equilibrium reactions simultaneously with a single iteration loop. Because CaSO4 is sparingly soluble, it is assumed to exist as a solid over the entire RH range to simplify the algorithm for calcium containing particles. Temperature-dependent mutual deliquescence relative humidity polynomials (valid from 240 to 310 K) for all the possible salt mixtures were constructed using the comprehensive Pitzer-Simonson-Clegg (PSC) activity coefficient model at 298.15 K and temperature-dependent equilibrium constants in MESA. Performance of MESA is evaluated for 16 representative mixed-electrolyte systems commonly found in tropospheric aerosols using PSC and two other multicomponent activity coefficient methods – Multicomponent Taylor Expansion Method (MTEM) of Zaveri et al. [2004], and the widely-used Kusik and Meissner method (KM), and the results are compared against the predictions of the Web-based AIM Model III or available experimental data. Excellent agreement was found between AIM, MESA-PSC, and MESA-MTEM predictions of the multistage deliquescence growth as a function of RH. On the other hand, MESA-KM displayed up to 20% deviations in the mass growth factors for common salt mixtures in the sulfate-poor cases while significant discrepancies were found in the predicted multistage