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Sample records for methanesulfonic acid monooxygenase

  1. Isolation and Characterization of Methanesulfonic Acid-Degrading Bacteria from the Marine Environment

    PubMed Central

    Thompson, A. S.; Owens, N.; Murrell, J. C.

    1995-01-01

    Two methylotrophic bacterial strains, TR3 and PSCH4, capable of growth on methanesulfonic acid as the sole carbon source were isolated from the marine environment. Methanesulfonic acid metabolism in these strains was initiated by an inducible NADH-dependent monooxygenase, which cleaved methanesulfonic acid into formaldehyde and sulfite. The presence of hydroxypyruvate reductase and the absence of ribulose monophosphate-dependent hexulose monophosphate synthase indicated the presence of the serine pathway for formaldehyde assimilation. Cell suspensions of bacteria grown on methanesulfonic acid completely oxidized methanesulfonic acid to carbon dioxide and sulfite with a methanesulfonic acid/oxygen stoichiometry of 1.0:2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and carbon dioxide for energy generation. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase. It was shown that methanol is not an intermediate in methanesulfonic acid metabolism, although these strains grew on methanol and other one-carbon compounds, as well as a variety of heterotrophic carbon sources. These two novel marine facultative methylotrophs have the ability to mineralize methanesulfonic acid and may play a role in the cycling of global organic sulfur. PMID:16535055

  2. Strongly Acidic Auxin Indole-3-Methanesulfonic Acid

    PubMed Central

    Cohen, Jerry D.; Baldi, Bruce G.; Bialek, Krystyna

    1985-01-01

    A radiochemical synthesis is described for [14C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [14C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles. PMID:16664007

  3. 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...

  4. Halogenated methanesulfonic acids: A new class of organic micropollutants in the water cycle.

    PubMed

    Zahn, Daniel; Frömel, Tobias; Knepper, Thomas P

    2016-09-15

    Mobile and persistent organic micropollutants may impact raw and drinking waters and are thus of concern for human health. To identify such possible substances of concern nineteen water samples from five European countries (France, Switzerland, The Netherlands, Spain and Germany) and different compartments of the water cycle (urban effluent, surface water, ground water and drinking water) were enriched with mixed-mode solid phase extraction. Hydrophilic interaction liquid chromatography - high resolution mass spectrometry non-target screening of these samples led to the detection and structural elucidation of seven novel organic micropollutants. One structure could already be confirmed by a reference standard (trifluoromethanesulfonic acid) and six were tentatively identified based on experimental evidence (chloromethanesulfonic acid, dichloromethanesulfonic acid, trichloromethanesulfonic acid, bromomethanesulfonic acid, dibromomethanesulfonic acid and bromochloromethanesulfonic acid). Approximated concentrations for these substances show that trifluoromethanesulfonic acid, a chemical registered under the European Union regulation REACH with a production volume of more than 100 t/a, is able to spread along the water cycle and may be present in concentrations up to the μg/L range. Chlorinated and brominated methanesulfonic acids were predominantly detected together which indicates a common source and first experimental evidence points towards water disinfection as a potential origin. Halogenated methanesulfonic acids were detected in drinking waters and thus may be new substances of concern. PMID:27267477

  5. Infrared studies of the reaction of methanesulfonic acid with trimethylamine on surfaces.

    PubMed

    Nishino, Noriko; Arquero, Kristine D; Dawson, Matthew L; Finlayson-Pitts, Barbara J

    2014-01-01

    Organosulfur compounds generated from a variety of biological as well as anthropogenic sources are oxidized in air to form sulfuric acid and methanesulfonic acid (MSA). Both of these acids formed initially in the gas phase react with ammonia and amines in air to form and grow new particles, which is important for visibility, human health and climate. A competing sink is deposition on surfaces in the boundary layer. However, relatively little is known about reactions after they deposit on surfaces. We report here diffuse reflectance infrared Fourier transform spectrometry (DRIFTS) studies of the reaction of MSA with trimethylamine (TMA) on a silicon powder at atmospheric pressure in synthetic air and at room temperature, either in the absence or in the presence of water vapor. In both cases, DRIFTS spectra of the product surface species are essentially the same as the transmission spectrum obtained for trimethylaminium methanesulfonate, indicating the formation of the salt on the surface with a lower limit to the reaction probability of γ > 10(-6). To the best of our knowledge, this is the first infrared study to demonstrate this chemistry from the heterogeneous reaction of MSA with an amine on a surface. This heterogeneous chemistry appears to be sufficiently fast that it could impact measurements of gas-phase amines through reactions with surface-adsorbed acids on sampling lines and inlets. It could also represent an additional sink for amines in the boundary layer, especially at night when the gas-phase reactions of amines with OH radical and ozone are minimized. PMID:24304088

  6. A cerium-lead redox flow battery system employing supporting electrolyte of methanesulfonic acid

    NASA Astrophysics Data System (ADS)

    Na, Zhaolin; Xu, Shengnan; Yin, Dongming; Wang, Limin

    2015-11-01

    A novel cerium-lead redox flow battery (RFB) employing Ce(IV)/Ce(III) and Pb(II)/Pb redox couples in the supporting electrolyte of methanesulfonic acid (MSA) is developed and preliminarily investigated. The RFB requires no additional catalyst and uses kinetically favorable reactions between low-cost reactants, and provides a desirable discharge voltage of approximately 1.7 V, with high average coulombic efficiency (CE) of 92% and energy efficiency (EE) of 86% over 800 cycles at 298 K. Stable cycling with an acceptable performance is achieved for a board operating temperature range of 253 K-313 K. The excellent performance obtained from the preliminary study suggests that the cerium-lead RFB promises to be applicable to large-scale energy storage for electricity grids.

  7. Surface and free tropospheric sources of methanesulfonic acid over the tropical Pacific Ocean

    SciTech Connect

    Zhang, Yuzhong; Wang, Yuhang; Gray, Burton A.; Gu, Dasa; Mauldin, L.; Cantrell, Chris; Bandy, Alan R.

    2014-07-28

    The production of sulfate aerosols through marine sulfur chemistry is critical to the climate system. However, not all sulfur compounds have been studied in detail. One such compound is methanesulfonic acid (MSA). In this study, we use a one-dimensional chemical transport model to analyze observed vertical profiles of gas-phase MSA during the Pacific Atmospheric Sulfur Experiment (PASE). The observed sharp decrease in MSA from the surface to 600m implies a surface source of 4.0×107 molecules/cm2/s. Evidence suggests that this source is photolytically enhanced. We also find that the observed large increase of MSA from the boundary layer into the lower free troposphere (1000-2000m) results mainly from the degassing of MSA from dehydrated aerosols. We estimate a source of 1.2×107 molecules/cm2/s through this pathway. This source of soluble MSA potentially provides an important precursor for new particle formation in the free troposphere over tropics, affecting the climate system through aerosol-cloud interactions.

  8. In vivo and in vitro antigenotoxic effect of nordihydroguaiaretic acid against SCEs induced by methyl methanesulfonate.

    PubMed

    Madrigal-Bujaidar, E; Díaz Barriga, S; Cassani, M; Márquez, P; Revuelta, P

    1998-11-01

    Nordihydroguaiaretic acid (NDGA) is a phenolic lignan which has shown to cause a variety of actions potentially useful for human health; therefore, in this investigation we determined its capacity for inhibiting the rate of sister chromatid exchanges (SCEs) induced by methyl methanesulfonate (MMS). We tested the effect of 0.25, 0.50, 1.0, and 2.0 microM of NDGA on the damage exerted by 55 microM of MMS. Cultured human lymphocytes from two female donors were used for the experiment. The best result concerning its modulatory action was obtained with 1.0 microM of NDGA; with this dose the mean inhibitory index including both donors reached 68.2%. The values obtained for the mitotic and proliferative indexes were not significantly modified with respect to the basal data. We also used the mouse bone marrow in vivo system to evaluate the inhibitory effect of the chemical. In this study we tested 1.0, 6.0, and 11.0 mg/kg of NDGA intraperitoneally (i.p.) administered 1 h before an i.p. injection of MMS (40 mg/kg). The best inhibitory index in this model corresponded to the dose of 11 mg/kg of NDGA (86.9%). The mitotic index and the average generation time showed no significant variation with respect to the control data. Our study established that NDGA produces antigenotoxic action in mammalian cells in vitro and in vivo. PMID:9804941

  9. Experimental and theoretical studies on methanesulfonic acid 1-methylhydrazide: Antimicrobial activities of its sulfonyl hydrazone derivatives

    NASA Astrophysics Data System (ADS)

    Özbek, Neslihan; Alyar, Saliha; Karacan, Nurcan

    2009-12-01

    Methanesulfonic acid 1-methylhydrazide ( msmh) and its sulfonyl hydrazone derivatives, salicylaldehyde- N-methylmethanesulfonylhydrazone ( salmsmh) and 2-hydroxy-1-naphthaldehyde- N-methylmethanesulfonylhydrazone ( nafmsmh) were synthesized and characterized by using FT-IR, 1H NMR, 13C NMR, LC-MS and elemental analysis. Conformation analysis of msmh based on DFT/B3LYP/6-311G(d) method was performed. 1H and 13C shielding tensors of msmh for the most stable conformer were calculated with GIAO/DFT/B3LYP/6-311++G(2d, 2p) methods in vacuo and various solvents such as DMSO, THF, acetonitrile, methanol and aqueous solution. The harmonic vibrational wavenumbers for the most stable conformer were calculated using at B3LYP/6-311G(d) level. Antimicrobial activity of the compounds was also screened against Gram-positive bacteria ( Staphylococcus aureus ATCC 25923, Bacillus cereus RSKK 863) and Gram-negative bacteria ( Escherichia coli ATCC 11230, Salmonella enterititis ATCC 40376, Pseudomonos aeruginosa ATCC 28753) by both disc diffusion and micro dilution methods.

  10. New Particle Formation and Growth from Methanesulfonic Acid, Amines, Water, and Organics

    NASA Astrophysics Data System (ADS)

    Arquero, K. D.; Ezell, M. J.; Finlayson-Pitts, B. J.

    2014-12-01

    Particles in the atmosphere can influence visibility, negatively impact human health, and affect climate. The largest uncertainty in determining global radiative forcing is attributed to atmospheric aerosols. While new particle formation in many locations is correlated with sulfuric acid in air, neither the gas-phase binary nucleation of H2SO4-H2O nor the gas-phase ternary nucleation of H2SO4-NH3-H2O alone can fully explain observations. An additional potential particle source, based on previous studies in this laboratory, is methanesulfonic acid (MSA) with amines and water vapor. However, organics are ubiquitous in the atmosphere, with secondary organic aerosol (SOA) being a major component of particles. Organics could be involved in the initial stages of particle formation by enhancing or inhibiting nucleation from sulfuric acid or MSA, in addition to contributing to their growth to form SOA. Experiments to measure the effects of a series of organics of varying structure on particle formation and growth from MSA, amines, and water were performed in a custom-built small volume aerosol flow tube reactor. Analytical instruments and techniques include a scanning mobility particle sizer to measure particle size distributions, sampling onto a weak cation exchange resin with analysis by ion chromatography to measure amine concentrations, and filter collection and analysis by ultra-high performance liquid chromatography tandem mass spectrometry to measure MSA concentrations. Organics were measured by atmospheric pressure chemical ionization tandem mass spectrometry. The impact of these organics on the initial particle formation as well as growth will be reported. The outcome is an improved understanding of fundamental chemistry of nucleation and growth to ultimately be incorporated into climate models to better predict how particles affect the global climate budget.

  11. Reactions of Methanesulfonic Acid with Amines and Ammonia as a Source of New Particles in Air.

    PubMed

    Chen, Haihan; Varner, Mychel E; Gerber, R Benny; Finlayson-Pitts, Barbara J

    2016-03-01

    New particle formation (NPF) from gaseous precursors as a significant source of aerosol needs to be better understood to accurately predict the impacts on visibility, climate change, and human health. While ternary nucleation of sulfuric acid, amines/NH3, and water is recognized as a significant driver for NPF, increasing evidence suggests a contribution from methanesulfonic acid (MSA) and amines under certain conditions. Here we report the formation of particles 2.5-10 nm in diameter from the reactions of MSA with methylamine (MA), dimethylamine (DMA), and NH3 at reaction times of 2.3-7.8 s in a flow reactor and compare these particles with those previously reported to be formed from reaction with trimethylamine (TMA). The effects of water vapor and concentrations of gaseous precursors on the particle number concentration and particle size were studied. The presence of water significantly enhances particle formation and growth. Under similar experimental conditions, particle number concentrations decrease in the order MA ≫ TMA ≈ DMA ≫ NH3, where NH3 is 2-3 orders of magnitude less efficient than DMA. Quantum chemical calculations of likely intermediate clusters were carried out to provide insights into the role of water and the different capacities of amines/NH3 in particle formation. Both gas-phase basicity and hydrogen-bonding capacity of amines/NH3 contribute to the potential for particles to form and grow. Our results indicate that, although amines typically have concentrations 1-3 orders of magnitude lower than that of NH3 in the atmosphere, they still play an important role in driving NPF. PMID:26379061

  12. Origin of dimethylsulfide, non-sea-salt sulfate, and methanesulfonic acid in eastern Antarctica

    NASA Astrophysics Data System (ADS)

    Cosme, E.; Hourdin, F.; Genthon, C.; Martinerie, P.

    2005-02-01

    Ignoring the origin of atmospheric chemicals is often a strong limitation to the full interpretation of their measurement. In this article, this question is addressed in the case of the sulfur species in Antarctica, with an original method of retrotransport of tracers. The retrotransport model is derived from the Laboratoire de Météorologie Dynamique Zoom-Tracers (LMD-ZT) atmospheric general circulation model, optimized for polar climate and expanded to simulate atmospheric sulfur chemistry. For two East Antarctic scientific stations (Dumont d'Urville and Vostok) the effects of transport and chemistry and the influence of oceanic, volcanic, and anthropogenic sources on dimethylsulfide (DMS), non-sea-salt (nss) sulfate, and methanesulfonic acid (MSA) concentrations are evaluated in summer and winter. The oceanic source largely dominates, but other sources can episodically be significant. The meridional origin and the age of DMS, MSA, and biogenic nss sulfate are also estimated. The latitudes of origin of MSA and nss sulfate are similar in summer, but they differ markedly in winter. This is a signature of their different chemical production scheme. Also, the interannual variability of the origin of the sulfur species at Vostok is weak compared to that at Dumont d'Urville. Acknowledging that the DMS concentrations in the ocean have no interannual variability in the model, this result suggests unsurprisingly that inland Antarctic stations may be better observation sites to monitor large-scale DMS bioproductivity variability than coastal sites are. The combination of slower chemistry and more intense atmospheric circulation in winter leads to unexpected results, such as a younger DMS in winter than in summer at Vostok.

  13. Post-depositional migration and preservation of methanesulfonic acid (MSA) in polar ice cores

    NASA Astrophysics Data System (ADS)

    Osman, M.; Marchal, O.; Guo, W.; Das, S. B.; Evans, M. J.

    2015-12-01

    Methanesulfonic acid (MSA; CH3SO3-) in ice cores is a unique, high-resolution proxy of regional sea ice behavior, marine primary productivity, and synoptic climatology. Significant uncertainties remain, however, in both our understanding of the production and transfer of MSA to the ice sheet, as well as its preservation over time, compromising the paleoclimatological utility of the proxy. Here we apply a numerical modeling approach to quantitatively investigate the post-depositional processes affecting MSA migration and preservation within the firn and ice column, building on recent observational and theoretical studies. Our model allows us to evaluate the timing and magnitude of the vertical movement of MSA in response to varying influences, including the competing effects of 1) concentration gradients of sea-salts typically deposited asynchronously to MSA, 2) snow accumulation and densification rates, and 3) in situ temperature gradients. We first test the model against a recently collected ice core from a high accumulation site in coastal West Antarctica, where monthly-resolved MSA records show an abrupt shift from a summer-to-winter maximum in MSA at ~23m depth (ρ ≈ 650 kg/m3), near the firn-ice transition. We find our model to be a robust predictor of the observed migrational features in this record, capturing both (i) the abrupt shift in summer-to-winter maximal concentrations of MSA (steady state ≈ 3.2 yrs), and (ii) the depression of the seasonal amplitude at depth. Further, our modeling results suggest post-depositional effects can lead to substantial interannual alteration of the MSA signal, contrary to previous assumptions that MSA migration is confined within annual layers at high accumulation sites. Using a broad range of polar MSA records and their associated, site-specific environmental conditions, we will evaluate the fidelity of subannual to interannual variability of MSA records and systematically determine the factors conducive to its

  14. Simplified mechanism for new particle formation from methanesulfonic acid, amines, and water via experiments and ab initio calculations

    PubMed Central

    Dawson, Matthew L.; Varner, Mychel E.; Perraud, Véronique; Ezell, Michael J.; Gerber, R. Benny; Finlayson-Pitts, Barbara J.

    2012-01-01

    Airborne particles affect human health and significantly influence visibility and climate. A major fraction of these particles result from the reactions of gaseous precursors to generate low-volatility products such as sulfuric acid and high-molecular weight organics that nucleate to form new particles. Ammonia and, more recently, amines, both of which are ubiquitous in the environment, have also been recognized as important contributors. However, accurately predicting new particle formation in both laboratory systems and in air has been problematic. During the oxidation of organosulfur compounds, gas-phase methanesulfonic acid is formed simultaneously with sulfuric acid, and both are found in particles in coastal regions as well as inland. We show here that: (i) Amines form particles on reaction with methanesulfonic acid, (ii) water vapor is required, and (iii) particle formation can be quantitatively reproduced by a semiempirical kinetics model supported by insights from quantum chemical calculations of likely intermediate clusters. Such an approach may be more broadly applicable in models of outdoor, indoor, and industrial settings where particles are formed, and where accurate modeling is essential for predicting their impact on health, visibility, and climate. PMID:23090988

  15. Simplified mechanism for new particle formation from methanesulfonic acid, amines, and water via experiments and ab initio calculations.

    PubMed

    Dawson, Matthew L; Varner, Mychel E; Perraud, Véronique; Ezell, Michael J; Gerber, R Benny; Finlayson-Pitts, Barbara J

    2012-11-13

    Airborne particles affect human health and significantly influence visibility and climate. A major fraction of these particles result from the reactions of gaseous precursors to generate low-volatility products such as sulfuric acid and high-molecular weight organics that nucleate to form new particles. Ammonia and, more recently, amines, both of which are ubiquitous in the environment, have also been recognized as important contributors. However, accurately predicting new particle formation in both laboratory systems and in air has been problematic. During the oxidation of organosulfur compounds, gas-phase methanesulfonic acid is formed simultaneously with sulfuric acid, and both are found in particles in coastal regions as well as inland. We show here that: (i) Amines form particles on reaction with methanesulfonic acid, (ii) water vapor is required, and (iii) particle formation can be quantitatively reproduced by a semiempirical kinetics model supported by insights from quantum chemical calculations of likely intermediate clusters. Such an approach may be more broadly applicable in models of outdoor, indoor, and industrial settings where particles are formed, and where accurate modeling is essential for predicting their impact on health, visibility, and climate. PMID:23090988

  16. 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

  17. Water structure at aqueous solution surfaces of atmospherically relevant dimethyl sulfoxide and methanesulfonic acid revealed by phase-sensitive sum frequency spectroscopy.

    PubMed

    Chen, Xiangke; Allen, Heather C

    2010-11-25

    Interfacial water structures of aqueous dimethyl sulfoxide (DMSO) and methanesulfonic acid (MSA) were studied by Raman, infrared, and conventional and phase-sensitive vibrational sum frequency generation (VSFG) spectroscopies. Through isotopic dilution, we probed bulk water hydrogen bonding strength using the vibrational frequency of dilute OD in H(2)O. As indicated by the frequency shift of the OD frequency, it is shown that DMSO has little influence on the average water hydrogen bonding strength at low concentrations in contrast with an overall weakening effect for MSA. For the water structure at the surface of aqueous solutions, although conventional VSFG spectra suggest only slight structural changes with DMSO and a red shift of hydrogen-bonded water OH frequency, phase-sensitive VSFG reveals more thoroughly structural changes in the presence of both DMSO and MSA. In the case of DMSO, reorientation of interfacial water molecules with their hydrogens pointing up toward the oxygen of the S=O group is observed. For MSA, the interfacial water structure is affected by both the dissociated methanesulfonate anions and the hydronium ions residing at the surface. Both the methanesulfonate anions and the hydronium ions have surface preference; therefore, the electric double layer (EDL) formed at the surface is relatively thin, which leads to partial reorientation of interface water molecules with net orientation of water hydrogens up. Surface DMSO molecules are more effective at reorienting surface water relative to MSA molecules. PMID:21047087

  18. 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

  19. EFFECTS OF ANESTHESIA (TRICAINE METHANESULFONATE, MS-222) BIOTRANSFORMATION IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

    EPA Science Inventory

    Tricaine methanesulfonate (3-aminobenzoic acid eithyl ester methanesulfonate, tricaine, MS-222, Finquel), an anesthetic for fish, has been used extensively in aquatic toxicology to allow surgical procedures for in vivo studies and to permit in vitro preparations of isolated perfu...

  20. Modulation of ultraviolet light-, ethyl methanesulfonate-, and 7,12-dimethylbenz(A)anthracene-induced unscheduled DNA synthesis by retinol and retinoic acid in the primary rat hepatocyte

    SciTech Connect

    Budroe, J.D.; Shaddock, J.G.; Casciano, D.A.

    1987-01-01

    The effects of retinol and retinoic acid on unscheduled DNA synthesis (UDS) in primary Sprague-Dawley rat hepatocytes were studied in the presence and absence of know chemical and physical mutagens. Neither retinol or retinoic acid caused a significant increase in UDS over solvent control at concentrations ranging from 1 ..mu..M to 50 ..mu..M. Retinol and retinoic acid did not significantly affect 200..mu..g/mL ethyl methanesulfonate (EMS)- or 32 J/m/sup 2/ ultraviolet light (UV)-induced UDS at concentrations ranging from 1..mu..M to 50 ..mu..M. In contrast, retinol and retinoic acid significantly inhibited 2.5 ..mu..g/mL and 5.0 ..mu..g/mL 7,12-dimethyl-benz(a)-anthracene(DMBA)-induced UDS at concentrations of 1..mu..M or greater. Retinol-and retinoic acid-induced hepatocytotoxicity was studied in vitro using lactate dehydrogenase (LDH) release as an indicator of cytoxicity. Neither retinol nor retinoic acid caused significant increases in LDH release over solvent control 3 hours after treatment, whereas retinol caused a biologically significant increase in LDH release 24 hours posttreatment at concentrations of 50 ..mu..M and 100 ..mu..M. These data suggest that nontoxic concentrations of retinol and retinoic acid do not inhibit the DNA excision repair process but apparently affect the effective DNA adduct load due to the ultimate species of DMBA metabolite responsible for hepatocellular DNA damage.

  1. Modulation of ultraviolet light-, ethyl methanesulfonate-, and 7,12-dimethylbenz(a)anthracene-induced unscheduled DNA synthesis by retinol and retinoic acid in the primary rat hepatocyte

    SciTech Connect

    Budroe, J.D.; Shaddock, J.G.; Casciano, D.A.

    1987-01-01

    The effects of retinol and retinoic acid on unscheduled DNA synthesis (UDS) in primary Sprague-Dawley rat hepatocytes were studied in the presence and absence of known chemical and physical mutagens. Neither retinol nor retinoic acid caused a significant increase in UDS over solvent control at concentrations ranging from 1 microM to 50 microM. Retinol and retinoic acid did not significantly affect 200 micrograms/mL ethyl methanesulfonate(EMS)- or 32 J/m2 ultraviolet light(UV)-induced UDS at concentrations ranging from 1 microM to 50 microM. In contrast, retinol and retinoic acid significantly inhibited 2.5 micrograms/mL and 5.0 micrograms/mL 7,12-dimethyl-benz(a)anthracene(DMBA)-induced UDS at concentrations of 1 microM or greater. Retinol- and retinoic acid-induced hepatocytotoxicity was studied in vitro using lactate dehydrogenase (LDH) release as an indicator of cytoxicity. Neither retinol nor retinoic acid caused significant increases in LDH release over solvent control 3 hours after treatment, whereas retinol caused a biologically significant increase in LDH release 24 hours posttreatment at concentrations of 50 microM and 100 microM. These data suggest that nontoxic concentrations of retinol and retinoic acid do not inhibit the DNA excision repair process but apparently affect the effective DNA adduct load due to the ultimate species of DMBA metabolite responsible for hepatocellular DNA damage.

  2. Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts.

    PubMed

    Wrobel, Katarzyna; Kannamkumarath, Sasi S; Wrobel, Kazimierz; Caruso, Joseph A

    2003-01-01

    In this work, the use of methanesulfonic acid for protein hydrolysis is proposed for evaluation of Se-methionine in yeast, Brazil nuts, and possibly other selenium-rich biological samples. The hydrolysis was carried out by heating the sample with 4 mol L(-1) acid at reflux for 8 h. Two chromatographic techniques (size-exclusion and ion-pairing) coupled with ICP-MS detection were used to compare the release of Se-methionine from proteins by enzymatic (proteinase K, protease XIV) and acid hydrolyses. A more efficient liberation of Se-methionine was observed by acid hydrolysis. For quantification, the sample extracts were introduced onto a C8 Alltima column, and the separation was achieved with a mobile phase containing 5 mmol L(-1) hexanesulfonic acid in citrate buffer (pH 4.5)/methanol (95:5). The results obtained by standard addition showed 816+/-17 micro g g(-1) and 36.2+/-1.5 micro g g(-1) of selenium in the form of Se-methionine in yeast and nuts, respectively (65% and 75% of total selenium). PMID:12520449

  3. (+)-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

  4. Behavioural Effects of the Commonly Used Fish Anaesthetic Tricaine Methanesulfonate (MS-222) on Zebrafish (Danio rerio) and Its Relevance for the Acetic Acid Pain Test

    PubMed Central

    Nordgreen, Janicke; Tahamtani, Fernanda M.; Janczak, Andrew M.; Horsberg, Tor Einar

    2014-01-01

    The pros and cons of using anaesthesia when handling fish in connection with experiments are debated. A widely adopted practice is to wait thirty minutes after anaesthesia before behavioural observations are initiated, but information about immediate effects of a treatment is then lost. This is pertinent for responses to acute stressors, such as acid injection in the acetic acid pain test. However, omission of anaesthetics in order to obtain data on immediate responses will compromise the welfare of fish and contribute to experimental noise due to stress. We therefore tested the effect of tricaine methanesulfonate on the behaviour of zebrafish. We predicted that tricaine (MS 222) would decrease swimming velocity and that the control fish would show an increased level of anxiety- and stress-related behaviours compared to the tricaine group. Following acclimatization to the test tank, baseline behaviour was recorded before immersion in either tricaine (168 mg l−1, treatment group, N = 8) or tank water (control group, N = 7). Latencies to lose equilibrium and to lose response to touch were registered. The fish was then returned to the test tank, and the latency to regain equilibrium was registered in anaesthetized fish. When equilibrium was regained, and at five, thirty and sixty minutes after the fish had been returned to the test tank, behaviour was recorded. The tricaine fish showed the following responses (mean ± sd): latency to lose equilibrium 22.6 s±3.9; latency to lose response to touch 101.9 s±26.8; latency to regain equilibrium 92.0 s±54.4. Contrary to our predictions, neither treatment caused a change in any of the behaviours registered. This indicates that tricaine has no effect on several commonly used behavioural parameters, and that it may be unnecessary to postpone behavioural observations to 30 min after anaesthesia. PMID:24658262

  5. Ice core sulfur and methanesulfonic acid (MSA) records from southern Greenland document North American and European air pollution and suggest a decline in regional biogenic sulfur emissions.

    NASA Astrophysics Data System (ADS)

    Pasteris, D. R.; McConnell, J. R.; Burkhart, J. F.; Saltzman, E. S.

    2014-12-01

    Sulfate aerosols have an important cooling effect on the Earth because they scatter sunlight back to space and form cloud condensation nuclei. However, understanding of the atmospheric sulfur cycle is incomplete, leading to uncertainty in the assessment of past, present and future climate forcing. Here we use annually resolved observations of sulfur and methanesulfonic acid (MSA) concentration in an array of precisely dated Southern Greenland ice cores to assess the history of sulfur pollution emitted from North America and Europe and the history of biogenic sulfate aerosol derived from the North Atlantic Ocean over the last 250 years. The ice core sulfur time series is found to closely track sulfur concentrations in North American and European precipitation since records began in 1965, and also closely tracks estimated sulfur emissions since 1850 within the air mass source region as determined by back trajectory analysis. However, a decline to near-preindustrial sulfur concentrations in the ice cores after 1995 that is not so extensive in the source region emissions indicates that there has been a change in sulfur cycling over the last 150 years. The ice core MSA time series shows a decline of 60% since the 1860s, and is well correlated with declining sea ice concentrations around Greenland, suggesting that the phytoplankton source of biogenic sulfur has declined due to a loss of marginal sea ice zone habitat. Incorporating the implied decrease in biogenic sulfur in our analysis improves the match between the ice core sulfur record and the source region emissions throughout the last 150 years, and solves the problem of the recent return to near-preindustrial levels in the Greenland ice. These findings indicate that the transport efficiency of sulfur air pollution has been relatively stable through the industrial era and that biogenic sulfur emissions in the region have declined.

  6. Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity.

    PubMed Central

    Katavic, V; Reed, D W; Taylor, D C; Giblin, E M; Barton, D L; Zou, J; Mackenzie, S L; Covello, P S; Kunst, L

    1995-01-01

    In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids. PMID:7784510

  7. Interaction of the antitumour drug 4'-(9-acridinylamino)-methanesulfon-m-anisidine.HCl (m-AMSA) with nucleic acids.

    PubMed Central

    Hudecz, F; Kajtár, J; Szekerke, M

    1981-01-01

    The interaction of AMSA with nucleic acids was studied by several techniques. Melting temperature and CD studies equally suggest that AMSA-binding is interfering with the secondary structure of DNA. An overlap by two mechanism of binding seems to exist. Based on the CD measurements at low drug concentration intercalation is the most likely way of binding. At higher drug concentration stacking interaction predominates leading to cooperativity and formation of oriented sheets of aromatic ring-systems as reflected in the optical activity induced in the metachromatic band of the achiral drug. No base-pair specificity could be confirmed; however, a high affinity of AMSA to poly(A) chains was demonstrated. The CD measurements did not indicate any significant interaction with RNA. The selectivity of the AMSA-DNA interaction can be regarded as an important argument in favour of the role of this interaction in the anti-tumour effect of the drug. PMID:6174949

  8. Synthesis and antiestrogenic activity of [3,4-dihydro-2-(4-methoxyphenyl)-1-naphthalenyl][4-[2-(1-pyrrolidinyl)ethoxy]-phenyl]methanone, methanesulfonic acid salt.

    PubMed

    Jones, C D; Suarez, T; Massey, E H; Black, L J; Tinsley, F C

    1979-08-01

    Acylation of the sodio anion of beta-tetralone with phenyl anisoate, followed by a Grignard reaction of the resultant 4 with 4-methoxyphenylmagnesium bromide, gave rise to two novel dihydronaphthalene isomers 5 and 6. Regioselective demethylation of either 5 or 6 by NaSEt produced [3,4-dihydro-2-(4-methoxyphenyl)-1-naphthalenyl](4-hydroxyphenyl)methanone (7). Etherification of the phenolic group of 7 by N-(2-chloroethyl)pyrrolidine and subsequent methanesulfonate salt formation provided [3,4-dihydro-2-(4-methoxyphenyl)-1-maphthalenyl

  9. Differences in metabolism of the marine biotoxin okadaic acid by human and rat cytochrome P450 monooxygenases.

    PubMed

    Kolrep, Franziska; Hessel, Stefanie; These, Anja; Ehlers, Anke; Rein, Kathleen; Lampen, Alfonso

    2016-08-01

    The ingestion of seafood contaminated with the marine biotoxin okadaic acid (OA) can lead to diarrhetic shellfish poisoning with symptoms like nausea, vomiting and abdominal cramps. Both rat and the human hepatic cytochrome P450 monooxygenases (CYP) metabolize OA. However, liver cell toxicity of metabolized OA is mainly unclear. The aim of our study was to detect the cellular effects in HepG2 cells exposed to OA in the presence of recombinant CYP enzymes of both rat and human for the investigation of species differences. The results should be set in correlation with a CYP-specific metabolite pattern. Comparative metabolite profiles of OA after incubation in rat and human recombinant CYP enzymes were established by using LC-MS/MS technique. Results demonstrated that metabolism of OA to oxygenated metabolites correlates with detoxification which was mainly catalyzed by human CYP3A4 and CYP3A5. Detoxification by rat Cyp3a1 was lower compared to human CYP3A enzymes and activation of OA by Cyp3a2 was observed, coincident with minor overall conversion capacity of OA. By contrast human and rat CYP1A2 seem to activate OA into cytotoxic intermediates. In conclusion, different mechanisms of OA metabolism may occur in the liver. At low OA doses, the human liver is likely well protected against cytotoxic OA, but for high shellfish consumers a potential risk cannot be excluded. PMID:26374342

  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. Evidence for a transient peroxynitro acid in the reaction catalyzed by nitronate monooxygenase with propionate 3-nitronate.

    PubMed

    Smitherman, Crystal; Gadda, Giovanni

    2013-04-16

    Nitronate monooxygenase is a flavin-dependent enzyme that catalyzes the denitrification of propionate 3-nitronate (P3N) and other alkyl nitronates. The enzyme was previously known as 2-nitropropane dioxygenase, until its reclassification in 2010 by the IUBMB. Physiologically, the monooxygenase from fungi protects the organism from the environmental occurrence of P3N, which shuts down the Krebs cycle by inactivating succinate dehydrogenase and fumarase. The inhibition of these enzymes yields severe neurological disorders or death. Here, we have used for the first time steady-state and rapid kinetics, viscosity and pH effects, and time-resolved absorbance spectroscopy of the enzyme in turnover with P3N and the substrate analogue ethyl nitronate (EN) to elucidate the mechanism of the reaction. A transient increase in absorbance at ∼300 nm, never reported before, was seen during steady-state turnover of the enzyme with P3N and oxygen, with no concomitant changes between 400 and 600 nm. The transient species was not detected when oxygen was absent. Anaerobic reduction of the enzyme with P3N yielded anionic flavosemiquinone and was fast (e.g., ≥1900 s(-1)). Steady-state kinetics demonstrated that oxygen reacts before the release of the product of P3N oxidation from the enzyme. No pH effects were seen with P3N on kcat/Km, kcat/Koxygen, and kcat; in contrast, with EN, the kcat/Km and kcat decreased with increasing pH defining two plateaus and a pKa ∼ 8.0. Solvent viscosity at the pH optima suggested product release as being partially controlling the overall rate of turnover with the physiological substrate and its analogue. A mechanism that satisfies the kinetic results is proposed. PMID:23530838

  13. Imino-Oxy Acetic Acid Dealkylation as Evidence for an Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed by Peptidylglycine α-Hydroxylating Monooxygenase (PHM)

    PubMed Central

    McIntyre, Neil R.; Lowe, Edward W.; Merkler, David J.

    2009-01-01

    Peptidylglycine α-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl α-carbon in a reaction that requires O2 and ascorbate. Subsequent dealkylation of the α-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a non-coupled, type II dicopper monooxygenase which activates O2 at only a single copper atom, CuM. In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The D(V/K)APPARENT, BIAA decreased from 14.7 ± 1.0 as [O2] → 0 to 1.0 ± 0.2 as [O2] → ∞ suggesting the dissociation rate constant from the PHM·BIAA complex decreases as [O2] increases; thereby, reducing the steady-state concentration of [PHM]free. BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked CuII-alkoxide intermediate with a quartet spin state responsible BIAA oxidation. PMID:19569683

  14. Colorimetric assay of noramidopyrine methanesulfonate sodium in formulations and in blood and urine samples.

    PubMed

    Diab, A H

    1977-04-01

    A simple, rapid, specific and sensitive colorimetric method is proposed for the quantitative estimation of noramido-pyrine methanesulfonate sodium in different dosage forms as well as in blood and urine samples. The method is based on the reaction of 3-sulfonic-5-amino-alpha-naphthol with formaldehyde liberated from noramidopyrine methanesulfonate sodium after treatment with conc. sulfuric acid where a yellow colour appeared immediately which turned to blue on dilution with water. The blue colour obeyed Beer's law (10--400 microgram) and remained stable for more than 1 h. The effect of other drugs, tablet excipients, parentral vehicles and suppository bases was studied. PMID:896911

  15. Methanesulfonate (MSA) Catabolic Genes from Marine and Estuarine Bacteria.

    PubMed

    Henriques, Ana C; De Marco, Paolo

    2015-01-01

    Quantitatively, methanesulfonate (MSA) is a very relevant compound in the global biogeochemical sulfur cycle. Its utilization by bacteria as a source of carbon and energy has been described and a specific enzyme, methanesulfonate monooxygenase (MSAMO), has been found to perform the first catabolic step of its oxidation. Other proteins seemingly involved in the import of MSA into bacterial cells have been reported. In this study, we obtained novel sequences of genes msmA and msmE from marine, estuary and soil MSA-degraders (encoding the large subunit of the MSAMO enzyme and the periplasmic component of the import system, respectively). We also obtained whole-genome sequences of two novel marine Filomicrobium strains, Y and W, and annotated two full msm operons in these genomes. Furthermore, msmA and msmE sequences were amplified from North Atlantic seawater and analyzed. Good conservation of the MsmA deduced protein sequence was observed in both cultured strains and metagenomic clones. A long spacer sequence in the Rieske-type [2Fe-2S] cluster-binding motif within MsmA was found to be conserved in all instances, supporting the hypothesis that this feature is specific to the large (α) subunit of the MSAMO enzyme. The msmE gene was more difficult to amplify, from both cultivated isolates and marine metagenomic DNA. However, 3 novel msmE sequences were obtained from isolated strains and one directly from seawater. With both genes, our results combined with previous metagenomic analyses seem to imply that moderate to high-GC strains are somehow favored during enrichment and isolation of MSA-utilizing bacteria, while the majority of msm genes obtained by cultivation-independent methods have low levels of GC%, which is a clear example of the misrepresentation of natural populations that culturing, more often than not, entails. Nevertheless, the data obtained in this work show that MSA-degrading bacteria are abundant in surface seawater, which suggests ecological

  16. 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.

  17. Amine-Amine Exchange in Aminium-Methanesulfonate Aerosols

    SciTech Connect

    Dawson, Matthew L.; Varner, Mychel E.; Perraud, Veronique M.; Ezell, Michael J.; Wilson, Jacqueline M.; Zelenyuk, Alla; Gerber, Robert B.; Finlayson-Pitts, Barbara J.

    2014-12-18

    Aerosol particles are ubiquitous in the atmosphere and have been shown to impact the Earth’s climate, reduce visibility, and adversely affect human health. Modeling the evolution of aerosol systems requires an understanding of the species and mechanisms involved in particle growth, including the complex interactions between particle- and gas-phase species. Here we report studies of displacement of amines (methylamine, dimethylamine or trimethylamine) in methanesulfonate salt particles by exposure to a different gas-phase amine, using a single particle mass spectrometer, SPLAT II. The variation of the displacement with the nature of the amine suggests that behavior is dependent on water in or on the particles. Small clusters of methanesulfonic acid with amines are used as a model in quantum chemical calculations to identify key structural elements that are expected to influence water uptake, and hence the efficiency of displacement by gas-phase molecules in the aminium salts. Such molecular-level understanding of the processes affecting the ability of gas-phase amines to displace particle-phase aminium species is important for modeling the growth of particles and their impacts in the atmosphere.

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

    PubMed Central

    Stepanova, Anna N.; Yun, Jeonga; Robles, Linda M.; Novak, Ondrej; He, Wenrong; Guo, Hongwei; Ljung, Karin; Alonso, Jose M.

    2011-01-01

    The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed. PMID:22108406

  19. Human flavin-containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria.

    PubMed

    Cashman, J R; Bi, Y A; Lin, J; Youil, R; Knight, M; Forrest, S; Treacy, E

    1997-08-01

    Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N

  20. 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

  1. Vibrational, XRD and (13)C NMR studies of DL-phenylglycinium methanesulfonate crystal.

    PubMed

    Wołoszyn, Łukasz; Ilczyszyn, Marek; Ilczyszyn, Maria M; Haupa, Karolina

    2016-11-01

    A new crystal formed by DL-phenylglycine and methanesulfonic acid (PGLYMS) was obtained and studied by X-ray diffraction, IR and Raman spectroscopy, solid state NMR and DSC methods. Additionally, theoretical computations for the phenylglycine cation were carried out (DFT/B3LYP/aug-cc-pVDZ). Our results show that PGLYMS does not exhibit any phase transitions and crystallizes in the P21/c space group of monoclinic system (Z=4). Detailed analysis of its structure and its IR, Raman and NMR spectra is presented. PMID:27289350

  2. Brahmarasayana protects against Ethyl methanesulfonate or Methyl methanesulfonate induced chromosomal aberrations in mouse bone marrow cells

    PubMed Central

    2012-01-01

    Background Ayurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Rasayana is one of the eight branches of Ayurveda which refers to rejuvenant therapy. It has been reported that rasayanas have immuno-modulatory, antioxidant and antitumor functions, however, the genotoxic potential and modulation of DNA repair of many rasayanas have not been evaluated. Methods The present study assessed the role of Brahmarasayana (BR) on Ethyl methanesulfonate (EMS)-and Methyl methanesulfonate (MMS)-induced genotoxicity and DNA repair in in vivo mouse test system. The mice were orally fed with BR (5 g or 8 mg / day) for two months and 24 h later EMS or MMS was given intraperitoneally. The genotoxicity was analyzed by chromosomal aberrations, sperm count, and sperm abnormalities. Results The results have revealed that BR did not induce significant chromosomal aberrations when compared to that of the control animals (p >0.05). On the other hand, the frequencies of chromosomal aberrations induced by EMS (240 mg / kg body weight) or MMS (125 mg / kg body weight) were significantly higher (p<0.05) to that of the control group. The treatment of BR for 60 days and single dose of EMS or MMS on day 61, resulted in significant (p <0.05) reduction in the frequency of chromosomal aberrations in comparison to EMS or MMS treatment alone, indicating a protective effect of BR. Constitutive base excision repair capacity was also increased in BR treated animals. Conclusion The effect of BR, as it relates to antioxidant activity was not evident in liver tissue however rasayana treatment was observed to increase constitutive DNA base excision repair and reduce clastogenicity. Whilst, the molecular mechanisms of such repair need further exploration, this is the first report to demonstrate these effects and provides further evidence for the role of brahmarasayana in the possible improvement of quality of life. PMID:22853637

  3. Positively charged amino acids are essential for electron transfer and protein-protein interactions in the soluble methane monooxygenase complex from Methylococcus capsulatus (Bath).

    PubMed

    Balendra, Suki; Lesieur, Claire; Smith, Thomas J; Dalton, Howard

    2002-02-26

    The soluble methane monooxygenase (sMMO) complex from Methylococcus capsulatus (Bath) catalyses oxygen- and NAD(P)H-dependent oxygenation of methane, propene, and other substrates. Whole-complex sMMO oxygenase activity requires all three sMMO components: the hydroxylase, the reductase, and protein B. Also, in the presence of hydrogen peroxide, the hydroxylase alone catalyzes substrate oxygenation via the peroxide shunt reaction. We investigated the effect of amine cross-linking on hydroxylase activity to probe the role of a gross conformational change that occurs in the hydroxylase upon binding of the other protein components. The cross-linker inhibited hydroxylase activity in the whole complex, but this effect was due to covalent modification of primary amine groups rather than cross-linking. Covalent modification of arginine side-chains on the hydroxylase had a similar effect, but, most remarkably, neither form of modification affected the activity of the hydroxylase via the peroxide shunt reaction. It was shown that covalent modification of positively charged groups on the hydroxylase, which occurred at multiple sites, interfered with its physical and functional interactions with protein B and with the passage of electrons from the reductase. These results indicate that protein B and the reductase of the sMMO complex interact via positively charged groups on the surface of the hydroxylase to induce a conformational change that is necessary for delivery of electrons into the active site of the hydroxylase. Modification of positively charged groups on protein B had no effect on its function, consistent with the hypothesis that positively charged groups on the hydroxylase interact with negative charges on protein B. Thus, we have discovered a means of specifically inactivating the interactions between the sMMO complex while preserving the catalytic activity of the hydroxylase active site which provides a new method of studying intercomponent interactions within s

  4. 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

  5. Oxygenases for aliphatic hydrocarbons and fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxygenases catalyzing the insertion of oxygen into either aliphatic hydrocarbons or fatty acids have great similarity. There are two classes of oxygenases: monooxygenases and dioxygenases. Dioxygenase inserts both atoms of molecular oxygen into a substrate, whereas monooxygenase incorporates one a...

  6. 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

  7. Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2.

    PubMed

    Yu, J; Chang, P K; Ehrlich, K C; Cary, J W; Montalbano, B; Dyer, J M; Bhatnagar, D; Cleveland, T E

    1998-12-01

    The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee. PMID:9835571

  8. 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

  9. 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

  10. 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.

  11. 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

  12. METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS

    EPA Science Inventory

    METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS. Geremy W. Knapp, Alan Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, Re...

  13. 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

  14. Capillary ion chromatography with on-column focusing for ultra-trace analysis of methanesulfonate and inorganic anions in limited volume Antarctic ice core samples.

    PubMed

    Rodriguez, Estrella Sanz; Poynter, Sam; Curran, Mark; Haddad, Paul R; Shellie, Robert A; Nesterenko, Pavel N; Paull, Brett

    2015-08-28

    Preservation of ionic species within Antarctic ice yields a unique proxy record of the Earth's climate history. Studies have been focused until now on two proxies: the ionic components of sea salt aerosol and methanesulfonic acid. Measurement of the all of the major ionic species in ice core samples is typically carried out by ion chromatography. Former methods, whilst providing suitable detection limits, have been based upon off-column preconcentration techniques, requiring larger sample volumes, with potential for sample contamination and/or carryover. Here, a new capillary ion chromatography based analytical method has been developed for quantitative analysis of limited volume Antarctic ice core samples. The developed analytical protocol applies capillary ion chromatography (with suppressed conductivity detection) and direct on-column sample injection and focusing, thus eliminating the requirement for off-column sample preconcentration. This limits the total sample volume needed to 300μL per analysis, allowing for triplicate sample analysis with <1mL of sample. This new approach provides a reliable and robust analytical method for the simultaneous determination of organic and inorganic anions, including fluoride, methanesulfonate, chloride, sulfate and nitrate anions. Application to composite ice-core samples is demonstrated, with coupling of the capillary ion chromatograph to high resolution mass spectrometry used to confirm the presence and purity of the observed methanesulfonate peak. PMID:26206628

  15. 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.

  16. 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

  17. 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

  18. A review of tricaine methanesulfonate for anesthesia of fish

    SciTech Connect

    Carter, Kathleen M.; Woodley, Christa M.; Brown, Richard S.

    2011-01-01

    Tricaine methanesulfonate (TMS) is the only FDA approved anesthetic for use in a select number of fish species, including salmonids. It is used widely in hatcheries and research to immobilize fish for marking or transport and to suppress sensory systems during invasive procedures. Improper use can decrease fish viability and possibly distort physiological data. Since animals may be anesthetized by junior staff or students who may have little experience in fish anesthesia, training in the proper use of TMS may decrease variability in results and increase fish survival. This document acts as a primer on the use of TMS for anesthetizing juvenile salmonids, with an emphasis on its use in surgical applications. Within, we briefly discuss many aspects TMS. We describe the legal uses for TMS, and what is currently known about the proper storage and preparation of the anesthetic. We outline methods and precautions for administration and changes in fish behavior during progressively deeper anesthesia. We also discuss the physiological effects of TMS and its potential for decreasing fish health.

  19. 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

  20. [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

  1. Complete Genome Sequences of Two Strains of “Candidatus Filomicrobium marinum,” a Methanesulfonate-Degrading Species

    PubMed Central

    Henriques, Ana C.

    2015-01-01

    Two novel methanesulfonate-degrading bacterial strains of “Candidatus Filomicrobium marinum” (strains Y and W) were isolated from a marine water enrichment, and their complete genome sequences are presented here. These are the first full genomes reported for the genus Filomicrobium and for methanesulfonate (MSA)-degrading bacteria. PMID:25953167

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Preliminary Method for Direct Quantification of Colistin Methanesulfonate by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

    PubMed Central

    Niece, Krista L.

    2015-01-01

    Colistin use has increased in response to the advent of infections caused by multidrug-resistant organisms. It is administered parenterally as an inactive prodrug, colistin methanesulfonate (CMS). Various formulations of CMS and labeling conventions can lead to confusion about colistin dosing, and questions remain about the pharmacokinetics of CMS. Since CMS does not have strong UV absorbance, current methods employ a laborious process of chemical conversion to colistin followed by precolumn derivatization to detect formed colistin by high-performance liquid chromatography. Here, we report a method for direct quantification of colistin methanesulfonate by attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR). PMID:26124160

  8. 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

  9. Influence of Tricaine Methanesulfonate on Streptococcus agalactiae vaccination of Nile tilapia (Oreochromis niloticus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Experiments were conducted to study the influence of tricaine methanesulfonate (MS-222) on blood glucose levels and percent cumulative survival of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae 30 days post-vaccination with S. agalactiae vaccine or sham-vaccination wit...

  10. Effects of metomindate hydrochloride and tricaine methanesulfonate on the short term cortisol response in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of metomidate hydrochloride and tricaine methanesulfonate (MS-222) on cortisol stress response of channel catfish, Ictalurus punctatus, were examined during 10 minutes of sedation. Channel catfish were assigned to three treatments: 1. Metomidate hydrochloride (12.5 mg/L), 2. MS-222 (100...

  11. 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

  12. 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

  13. Refractory status epilepticus, serious rhabdomyolysis, acute liver injury, and pancytopenia after a massive intake of ethyl methanesulfonate: a case report

    PubMed Central

    Yamazaki, Hiroyuki; Tajima, Shogo; Takeuchi, Takahiro

    2015-01-01

    Ethyl methanesulfonate is a mutagenic, alkylating agent and considered harmful to humans at levels greater than a certain threshold; however, the toxicity at high doses remains unclear. We report a case of a Japanese man who presented with status epilepticus, rhabdomyolysis, pancytopenia, and hair loss after accidental ingestion of a massive amount of ethyl methanesulfonate. The patient completely recovered with critical care, including multiple antiepileptic drugs, renal replacement therapy, blood transfusion, granulocyte colony-stimulating factor therapy, and antibacterial/fungal prophylaxis. The case indicates that ethyl methanesulfonate causes neurotoxicity, hepatotoxicity, hematotoxicity, and renal toxicity, which can be successfully treated with appropriate palliative therapies. PMID:26629236

  14. 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

  15. 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

  16. Evaluation of organic acids as fuel cell electrolytes

    SciTech Connect

    Ahmad, J.; Nguyen, T.H.; Foley, R.T.

    1981-11-01

    The electrochemical behavior of methanesulfonic acid, ethanesulfonic acid, and sulfoacetic acid as fuel cell electrolytes was studied in half-cell at various temperatures. The rate of the electro-oxidation of hydrogen at 115/degree/C was very high in methanesulfonic acid. The rate of the electro-oxidation of propane in all three acids was low even at 135/degree/C. Further, there is evidence for adsorption of these acids on the platinum electrode. It is concluded that anhydrous sulfonic acids are not good electrolytes; water solutions are required. Sulfonic acids containing unprotected carbon-hydrogen bonds are adsorbed on platinum and probably decompose during electrolysis. 9 refs.

  17. 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

  18. 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.

  19. endo-3,3-Dimethyl-4-oxobicyclo-[3.1.0]hexan-2-yl methane-sulfonate.

    PubMed

    Kremer, Adrian; Norberg, Bernadette; Krief, Alain; Wouters, Johan

    2010-01-01

    The relative configuration of the endo isomer of the title compound, C(9)H(14)O(4)S, has been established and the conformation of the diastereoisomer is discussed. The five-membered ring adopts an envelope conformation. The conformation of the methane-sulfonate substituent is stabilized by inter-molecular C-H⋯O hydrogen bonds. The crystal packing results in alternating layers of polar methane-sulfonates and stacked bicyclo-hexa-nyl rings parallel to ab. PMID:21580752

  20. 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

  1. 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

  2. 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

  3. Antimutagenic Effect of Dioscorea Pentaphylla on Genotoxic Effect Induced By Methyl Methanesulfonate in the Drosophila Wing Spot Test

    PubMed Central

    Prakash, G.; Hosetti, B. B.; Dhananjaya, B. L.

    2014-01-01

    Objectives: Plants as dietary sources are known to have several chemoprotective agents. Dioscorea pentaphylla is an important medicinal plant, which is often used as edible food. This study was undertaken to evaluate the antigenotoxic potential of D. pentaphylla extracts on the genotoxic effect induced by methyl methanesulfonate (MMS) in the Drosophila wing spot test. Materials and Methods: The somatic mutation and recombination test (SMART) was carried out in Drosophila melanogaster. In transheterogyous larvae, multiple wing hair (mwh 3-0.3) and flare (flr3-38.8) genes were used as markers of the extent of mutagenicity. Results: It was observed thatall the three extracts (petroleum ether, choloroform, and ethyl alcohol) in the combined treatment had significantly inhibited the effect of MMS-induced genotoxic effects. When compared to others, the ethanol extract showed a very significant antimutagenic activity. Conclusion: The compounds that are present in the extracts may directly interact with the methyl radical groups of MMS and inactivate them by chemical reaction. It is also possible that the compounds in the extract compete to interact with the nucleophilic sites in deoxyribonucleic acid (DNA), thus altering the binding of the mutagen to these sites. Although our results indicate that the compounds present in the extracts may directly interact with the methyl radical groups of MMS and inactivate them by chemical reaction, it may also be quite interesting to investigate through the other different mechanisms by which D. pentaphylla could interfere in vivo on the effect of genotoxic agents. PMID:25948963

  4. Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio).

    PubMed

    Wilson, Jolaine M; Bunte, Ralph M; Carty, Anthony J

    2009-11-01

    Despite the progressively increasing use of zebrafish (Danio rerio) in research, the most humane method of euthanasia for these fish has not been determined. Contemporary guidance documents state that hypothermia is an unacceptable method of euthanasia. The goal of this study was to compare rapid cooling and tricaine methanesulfonate (MS222) for zebrafish euthanasia. Zebrafish (n = 46) were euthanized by immersion in either an ice-water (4 degrees C or less) bath or unbuffered MS222 solution (250 mg/L; 25 to 30 degrees C). Another cohort (n = 10) was exposed to buffered MS222 to determine whether the acidity of the water alone caused distress. The times from exposure until the animals became unable to swim, right themselves, and death were measured, and signs of distress were recorded. Fish then were placed in a 'recovery tank' of system water to verify that recovery did not occur. Tissues were examined histologically. The mean time for euthanasia was longer and the number of fish exhibiting signs of distress was greater for fish exposed to MS222 than those exposed to chilled water. In addition, 4 of the 23 fish exposed to MS222 regained consciousness in the recovery tank, whereas none of 23 fish exposed to chilled water recovered. No histopathologic changes or evidence of ice crystal formation were seen in either group. In light of the faster time to death and fewer signs of distress in zebrafish euthanized by rapid cooling, we advocate this method as a humane veterinary practice. PMID:19930828

  5. 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

  6. 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.

  7. Induction of innate immune gene expression following methyl methanesulfonate-induced DNA damage in sea urchins.

    PubMed

    Reinardy, H C; Chapman, J; Bodnar, A G

    2016-02-01

    Sea urchins are noted for the absence of neoplastic disease and represent a novel model to investigate cellular and systemic cancer protection mechanisms. Following intracoelomic injection of the DNA alkylating agent methyl methanesulfonate, DNA damage was detected in sea urchin cells and tissues (coelomocytes, muscle, oesophagus, ampullae and gonad) by the alkaline unwinding, fast micromethod. Gene expression analyses of the coelomocytes indicated upregulation of innate immune markers, including genes involved in NF-κB signalling. Results suggest that activation of the innate immune system following DNA damage may contribute to the naturally occurring resistance to neoplastic disease observed in sea urchins. PMID:26911343

  8. The first Greenland ice core record of methanesulfonate and sulfate over a full glacial cycle

    SciTech Connect

    Hansson, M.E.; Saltzman, E.S. )

    1993-06-18

    The authors report on methanesulfonate and non-seasalt sulfate found in an artic ice core from Greenland. The ice core record stretches back in time roughly 130,000 years, through a full glacial cycle. This record reveals a decreasing concentration of MSA with the advance of the glacial period, and a drop in temperatures, while the non-seasalt sulfate increased in concentration. The MSA data is in contrast to similar measurements from the southern hemisphere. The ratio of MSA to non-seasalt sulfate is found to have a strong linear relationship to the temperature, higher ratios being associated with warmer climatic periods.

  9. Intra-Pleural Colistin Methanesulfonate Therapy for Pleural Infection caused by Carbapenem-Resistant Acinetobacter Baumannii: A Successful Case Report

    PubMed Central

    Rana, Muhammad Asim; Rahman, Basheer Abd El; Mady, Ahmed Fouad; Odat, Mohammed Al; AlHarthy, Abdurehman; Ramadan, Omar El Sayed; Mumtaz, Shahzad Ahmed; Omrani, Ali S.

    2014-01-01

    Infections caused by carbapenem-resistant, Gram-negative bacteria are an increasing clinical challenge, since the antimicrobial treatment options are often limited to colistin methanesulfonate. No data are available regarding the pharmacokinetics of colistin in pleural fluid. We report the case of a 92-year old man with ventilator-associated pneumonia and pleurisy caused by Acinetobacter baumannii and Escherichia coli, which were both multidrug-resistant. After an unsuccessful treatment with intravenous colistin methanesulfonate and imipen-em-cilastatin, the addition of intra-pleural colistin methanesulfonate to the intravenous treatment led to a prompt clinical, radiological and microbiological resolution. This is the first report of a successful use of intra-pleural colistin in the literature. The intra-pleural colistin therapy should be considered in selected cases of pleurisy caused by multi-resistant Gram-negative bacteria. PMID:25276329

  10. 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.

  11. Inhibition of mutagenicity in Salmonella typhimurium by Glycyrrhiza glabra extract, glycyrrhizinic acid, 18 alpha- and 18 beta-glycyrrhetinic acids.

    PubMed

    Zani, F; Cuzzoni, M T; Daglia, M; Benvenuti, S; Vampa, G; Mazza, P

    1993-12-01

    The effects of Glycyrrhiza glabra L. extract, glycyrrhizinic acid, 18 alpha- and 18 beta-glycyrrhetinic acids on the mutagenicity of the ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and ribose-lysine Maillard model systems were investigated by using the Salmonella/microsome reversion assay. The protocol used allowed us to detect desmutagenic and antimutagenic activity and to avoid false positive results due to toxicity. For all the compounds tested, no desmutagenic activity was observed against ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; only Glycyrrhiza glabra extract showed antimutagenic activity against ethyl methanesulfonate. On using the ribose-lysine mutagenic browning mixture, the desmutagenic activities of the Glycyrrhiza glabra extract, glycyrrhizinic acid, 18 alpha- and 18 beta-glycyrrhetinic acids were observed. 18 beta-Glycyrrhetinic acid was the most active compound. Glycyrrhiza glabra extract also exhibited antimutagenic activity against ribose-lysine. PMID:8302947

  12. 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.

  13. 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

  14. Surface and airborne measurements of organosulfur and methanesulfonate over the western United States and coastal areas

    NASA Astrophysics Data System (ADS)

    Sorooshian, Armin; Crosbie, Ewan; Maudlin, Lindsay C.; Youn, Jong-Sang; Wang, Zhen; Shingler, Taylor; Ortega, Amber M.; Hersey, Scott; Woods, Roy K.

    2015-08-01

    This study reports on ambient measurements of organosulfur (OS) and methanesulfonate (MSA) over the western United States and coastal areas. Particulate OS levels are highest in summertime and generally increase as a function of sulfate (a precursor) and sodium (a marine tracer) with peak levels at coastal sites. The ratio of OS to total sulfur is also highest at coastal sites, with increasing values as a function of normalized difference vegetation index and the ratio of organic carbon to elemental carbon. Correlative analysis points to significant relationships between OS and biogenic emissions from marine and continental sources, factors that coincide with secondary production, and vanadium due to a suspected catalytic role. A major OS species, methanesulfonate (MSA), was examined with intensive field measurements, and the resulting data support the case for vanadium's catalytic influence. Mass size distributions reveal a dominant MSA peak between aerodynamic diameters of 0.32-0.56 µm at a desert and coastal site with nearly all MSA mass (≥84%) in submicrometer sizes; MSA:non-sea-salt sulfate ratios vary widely as a function of particle size and proximity to the ocean. Airborne data indicate that relative to the marine boundary layer, particulate MSA levels are enhanced in urban and agricultural areas and also the free troposphere when impacted by biomass burning. Some combination of fires and marine-derived emissions leads to higher MSA levels than either source alone. Finally, MSA differences in cloud water and out-of-cloud aerosol are discussed.

  15. Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway.

    PubMed

    Jiang, Ying; Shan, Shigang; Chi, Linfeng; Zhang, Guanglin; Gao, Xiangjing; Li, Hongjuan; Zhu, Xinqiang; Yang, Jun

    2016-03-01

    Methyl methanesulfonate (MMS) is an alkylating agent that can induce cell death through apoptosis and necroptosis. The molecular mechanisms underlying MMS-induced apoptosis have been studied extensively; however, little is known about the mechanism for MMS-induced necroptosis. Therefore, we first established MMS-induced necroptosis model using human lung carcinoma A549 cells. It was found that, within a 24-h period, although MMS at concentrations of 50, 100, 200, 400, and 800 μM can induce DNA damage, only at higher concentrations (400 and 800 μM) MMS treatment lead to necroptosis in A549 cells, as it could be inhibited by the specific necroptotic inhibitor necrostatin-1, but not the specific apoptotic inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk). MMS-induced necroptosis was further confirmed by the induction of the necroptosis biomarkers including the depletion of cellular NADH and ATP and leakage of LDH. This necroptotic cell death was also concurrent with the increased expression of p53, p53-induced gene 3 (PIG-3), high mobility group box-1 protein (HMGB1), and receptor interaction protein kinase (RIP) but not the apoptosis-associated caspase-3 and caspase-9 proteins. Elevated reactive oxygen species (ROS) level was also involved in this process as the specific ROS inhibitor (4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC)) can inhibit the necroptotic cell death. Interestingly, knockdown of PIG-3 expression by small interfering RNA (siRNA) treatment can inhibit the generation of ROS. Taken together, these results suggest that MMS can induce necroptosis in A549 cells, probably through the PIG-3-ROS pathway. PMID:26472723

  16. 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

  17. Efficacy of metomidate and tricaine methanesulfonate to modulate the short-term cortisol stress response in channel catfish Ictalurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of the anesthetics metomidate and tricaine methanesulfonate to mitigate the cortisol stress response of channel catfish Ictalurus punctatus was evaluated during a 10 min confinement stress. Channel catfish (11.9 ± 0.5 g; mean ± SE) were transferred from holding tanks to confinement buck...

  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. Inhibition of excision-repair of ultraviolet damage in human cells by exposure to methyl methanesulfonate.

    PubMed

    Park, S D; Choi, K H; Hong, S W; Cleaver, J E

    1981-07-01

    Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2 mM), but repair of MMS damage was not inhibited by UV light. Because the pathways for excision of pyrimidine dimers and alkylation damage have previously been shown to be different, this observation implies a direct effect of alkylation on repair enzymes. We estimate that if inhibition is due to protein alkylation, the UV repair system must present an extremely large target to alkylation and may involve a complex of protein subunits in the order of 1 million daltons such that 1 or more alkylations occur per complex at the concentrations used. These results also indicate that the method of exposing cells to 2 DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves. PMID:7196494

  20. Surface and Airborne Measurements of Organosulfur and Methanesulfonate Over the Western United States and Coastal Areas

    PubMed Central

    Sorooshian, Armin; Crosbie, Ewan; Maudlin, Lindsay C.; Youn, Jong-Sang; Wang, Zhen; Shingler, Taylor; Ortega, Amber M.; Hersey, Scott; Woods, Roy K.

    2015-01-01

    This study reports on ambient measurements of organosulfur (OS) and methanesulfonate (MSA) over the western United States and coastal areas. Particulate OS levels are highest in summertime, and generally increase as a function of sulfate (a precursor) and sodium (a marine tracer) with peak levels at coastal sites. The ratio of OS to total sulfur (TS) is also highest at coastal sites, with increasing values as a function of Normalized Difference Vegetation Index (NDVI) and the ratio of organic carbon to elemental carbon. Correlative analysis points to significant relationships between OS and biogenic emissions from marine and continental sources, factors that coincide with secondary production, and vanadium due to a suspected catalytic role. A major OS species, methanesulfonate (MSA), was examined with intensive field measurements and the resulting data support the case for vanadium’s catalytic influence. Mass size distributions reveal a dominant MSA peak between aerodynamic diameters of 0.32—0.56 μm at a desert and coastal site with nearly all MSA mass (≥ 84%) in sub-micrometer sizes; MSA:non-sea salt sulfate ratios vary widely as a function of particle size and proximity to the ocean. Airborne data indicate that relative to the marine boundary layer, particulate MSA levels are enhanced in urban and agricultural areas, and also the free troposphere when impacted by biomass burning. Some combination of fires and marine-derived emissions leads to higher MSA levels than either source alone. Finally, MSA differences in cloud water and out-of-cloud aerosol are discussed. PMID:26413434

  1. Isolation of Two Novel Marine Ethylene-Assimilating Bacteria, Haliea Species ETY-M and ETY-NAG, Containing Particulate Methane Monooxygenase-like Genes

    PubMed Central

    Suzuki, Toshihiro; Nakamura, Takamichi; Fuse, Hiroyuki

    2012-01-01

    Two novel ethylene-assimilating bacteria, strains ETY-M and ETY-NAG, were isolated from seawater around Japan. The characteristics of both strains were investigated, and phylogenetic analyses of their 16S rRNA gene sequences showed that they belonged to the genus Haliea. In C1–4 gaseous hydrocarbons, both strains grew only on ethylene, but degraded ethane, propylene, and propane in addition to ethylene. Methane, n-butane, and i-butane were not utilized or degraded by either strain. Soluble methane monooxygenase-type genes, which are ubiquitous in alkene-assimilating bacteria for initial oxidation of alkenes, were not detected in these strains, although genes similar to particulate methane monooxygenases (pMMO)/ammonia monooxygenases (AMO) were observed. The phylogenetic tree of the deduced amino acid sequences formed a new clade near the monooxygenases of ethane-assimilating bacteria similar to other clades of pMMOs in type I, type II, and Verrucomicrobia methanotrophs and AMOs in alpha and beta proteobacteria. PMID:22307463

  2. 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

  3. 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

  4. Simultaneous determination of 13 quinolones in eggs using column high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry and depletion of pefloxacin methanesulfonate in eggs.

    PubMed

    Shen, Jianzhong; Li, Haiyan; Jiang, Haiyang; Zhou, Degang; Xu, Fei; Li, Jiancheng; Ding, Shuangyang

    2008-01-01

    An efficient method was developed for simultaneous determination of 13 quinolones--namely, enoaxacin (ENO), marbofloxacin (MAR), ciprofloxacin (CIP), norfloxacin (NOR), ofloxacin (OFL), pefloxacin methanesulfonate (PEF), danofloxacin (DAN), enrofloxacin (ENR), lomefloxacin (LOM), difloxacin (DIF), sarafloxacin (SAR), oxolinic acid (OXO), and flumequine (FLU)--in eggs by column liquid chromatography/electrospray ionization-tandem mass spectrometry. Samples were extracted with a phosphoric acid-phosphate buffer followed by purification with a solid-phase extraction cartridge. Recoveries for the 13 quinolones were 67-93% with intraday and interday coefficients of variation ranging from 4 to 9% and 2 to 18%, respectively. The limit of determination was 0.05 microg/kg for OXO and FLU; 0.1 microg/kg for MAR, OFL, CIP, LOM, DAN, SAR, DIF, NOR, and ENR; and 0.2 microg/kg for ENO and PEF. The method was also applied to study the depletion of PEF in eggs. The concentration of PEF increased and reached a maximum value on the third day, and then decreased rapidly until it could not be detected on day 32; its metabolite NOR was detectable on the second day, and then reached a maximum on the sixth day, after which it could not be detected until day 15. PMID:19202815

  5. 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

  6. 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

  7. 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.

  8. 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

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

    PubMed

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

    2015-02-12

    We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system. PMID:25590515

  10. Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate.

    PubMed Central

    Paine, A J

    1976-01-01

    1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system. PMID:962887

  11. Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate.

    PubMed

    Paine, A J

    1976-07-15

    1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system. PMID:962887

  12. 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

  13. 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

  14. Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics.

    PubMed

    Evenson, D P; Jost, L K; Baer, R K

    1993-01-01

    Effects of methyl methanesulfonate (MMS) on mouse testicular cell kinetics and sperm chromatin structure were determined flow cytometrically. Mice were exposed to a single ip injection of saline containing 0 or 150 mg/kg MMS. Relative ratios of 1N, 2N and 4N testicular cells were not affected until 22 days postexposure. Ratios of 1N cell types were altered from 13 to 22 days and were near normal by 25 days. This study revealed an MMS induced alteration of chromatin structure in testicular, elongated spermatids by the sperm chromatin structure assay (SCSA), a flow cytometric measure of the susceptibility of acridine orange stained sperm DNA to denaturation in situ. The SCSA also detected alterations in cauda sperm chromatin structure at 3 days, which was 8 days prior to alterations in sperm head morphology, indicating the increased sensitivity of the SCSA. SCSA data were practically similar whether measuring either fresh or frozen/thawed sperm, or whether measured by two different types of flow cytometers: a) laser driven, orthogonal optical axis; or b) low cost mercury arc lamp system with epiillumination. The data support the model of Sega and Owens [Mutat Res 111:227-244:1983] that MMS alkylates cysteine-SH groups in sperm protamines, thereby destabilizing sperm chromatin structure and leading to broken chromosomes and mutations. PMID:8444143

  15. Molecular Analysis of Mutations Induced in the Vermilion Gene of Drosophila Melanogaster by Methyl Methanesulfonate

    PubMed Central

    Nivard, MJM.; Pastink, A.; Vogel, E. W.

    1992-01-01

    The nature of DNA sequence changes induced by methyl methanesulfonate (MMS) at the vermilion locus of Drosophila melanogaster was determined after exposure of postmeiotic male germ cell stages. MMS is a carcinogen with strong preference for base nitrogen alkylation (s = 0.86). The spectrum of 40 intralocus mutations was dominated by AT -> GC transitions (23%), AT -> TA transversions (54%) and deletions (14%). The small deletions were preferentially found among mutants isolated in the F(1) (8/18), whereas the AT -> GC transitions exclusively occurred in the F(2) (6/22). The MMS-induced transversions and deletions are presumably caused by N-methyl DNA adducts, which may release apurinic intermediates, known to be a time-related process. Furthermore, MMS produces multilocus deletions, i.e., at least 30% of the F(1) mutants analyzed were of this type. A comparison of the mutational spectra of MMS with that produced by ethylnitrosourea (ENU), also in the vermilion locus of Drosophila, reveals major differences: predominantly transition mutations (61% GC -> AT and 18% AT -> GC) were found in both the F(1) and F(2) spectrum induced by ENU. It is concluded that the mutational spectrum of MMS is dominated by nitrogen DNA adducts, whereas with ENU DNA sequence changes mainly arose from modified oxygen in DNA. PMID:1628810

  16. Effects of anesthesia (tricaine methanesulfonate, MS222) on liver biotransformation in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Kolanczyk, Richard C; Fitzsimmons, Patrick N; McKim, James M; Erickson, Russell J; Schmieder, Patricia K

    2003-07-16

    The effect of tricaine methanesulfonate (MS222) on rainbow trout liver biotransformation rates was investigated with a microsomal model; an in vitro preparation that can be employed with or without the use of an anaesthetic. Two experimental sets of rainbow trout microsomes were tested; one representing in vivo or surgical tricaine exposures and the other representing in vitro tissue/organ collection tricaine exposures. Microsomal incubations were performed on these two experimental groups with phenol as substrate to assess the effects of tricaine on Phase I (ring-hydroxylation) and II (glucuronidation) liver biotransformation by monitoring production of hydroquinone (HQ), catechol (CAT), and phenylglucuronide (PG). The use of a 2-h 100 mg/l exposure of tricaine for surgical anesthesia with or without 24-h recovery did not significantly (P< or =0.05) affect rates of phenol (Phase I and II) biotransformation rates; nor, did the 5-min 300 mg/l tricaine exposure for isolated organ/tissue collection significantly (P< or =0.05) affect phenol (Phase I and II) biotransformation rates. There were also no significant statistical differences (P< or =0.05) in P450 protein levels, or 7-ethoxyresorufin-O-deethylase (EROD) activity in these microsomal assays between any of the tricaine treated rainbow trout and controls. PMID:12799110

  17. Interaction of Colistin and Colistin Methanesulfonate with Liposomes: Colloidal Aspects and Implications for Formulation

    PubMed Central

    WALLACE, STEPHANIE J.; LI, JIAN; NATION, ROGER L.; PRANKERD, RICHARD J.; BOYD, BEN J.

    2012-01-01

    Interaction of colistin and colistin methanesulfonate (CMS) with liposomes has been studied with the view to understanding the limitations to the use of liposomes as a more effective delivery system for pulmonary inhalation of this important class of antibiotic. Thus, in this study, liposomes containing colistin or CMS were prepared and characterized with respect to colloidal behavior and drug encapsulation and release. Association of anionic CMS with liposomes induced negative charge on the particles. However, degradation of the CMS to form cationic colistin over time was directly correlated with charge reversal and particle aggregation. The rate of degradation of CMS was significantly more rapid when associated with the liposome bilayer than when compared with the same concentration in aqueous solution. Colistin liposomes carried positive charge and were stable. Encapsulation efficiency for colistin was approximately 50%, decreasing with increasing concentration of colistin. Colistin was rapidly released from liposomes on dilution. Although the studies indicate limited utility of colistin or CMS liposomes for long duration controlled-release applications, colistin liposomes were highly stable and may present a potential opportunity for coformulation of colistin with a second antibiotic to colocalize the two drugs after pulmonary delivery. PMID:22623044

  18. The Response of Gray Treefrogs to Anesthesia by Tricaine Methanesulfonate (TMS or MS-222)

    PubMed Central

    Paduano, Mary; Colafrancesco, Kaitlen C.; Wong, Sarah A.; Caldwell, Michael S.; Gridi-Papp, Marcos

    2014-01-01

    The design of anesthetic protocols for frogs is commonly hindered by lack of information. Results from fishes and rodents do not always apply to frogs, and the literature in anurans is concentrated on a few species. We report on the response of treefrogs (Hyla chrysoscelis and H. versicolor) to tricaine methanesulfonate. Body mass did not differ significantly between the species or between sexes. In the first exposure of a frog to TMS, variation in induction time was best explained by species (H. chrysoscelis resisted longer) and body mass (larger animals resisted longer). Multiple exposures revealed a strong effect of individual variation on induction time and a significant increase of induction time with number of previous anesthesia events within the same day. Recovery time was mostly explained by individual variation, but it increased with total time in anesthetic and decreased with induction time. It also increased with number of days since the last series of anesthesias and decreased with number of previous uses of the anesthetic bath. This is one of the first studies of anesthesia in hylids and also one of the first assessments of the factors that influence the variability of the response to anesthesia within a species. PMID:24851186

  19. Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family†

    PubMed Central

    Sobrado, Pablo; Fitzpatrick, Paul F.

    2006-01-01

    The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of l-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402, 24–30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the Kd value for tryptophan or the Ki value for the competitive inhibitor indoleacetamide, and increases the Ki value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5- and 2-fold, respectively, and increases the Kd value for tryptophan and the Ki value for indolepyruvate by 31- and 17-fold, respectively, while having an only 2-fold effect on the Ki value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/Ktrp values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis. PMID:14636049

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Pharmacokinetics of Colistin in Cerebrospinal Fluid after Intraventricular Administration of Colistin Methanesulfonate

    PubMed Central

    Cusato, Maria; Accetta, Giovanni; Marinò, Valeria; Procaccio, Francesco; Del Gaudio, Alfredo; Iotti, Giorgio A.; Regazzi, Mario

    2012-01-01

    Intraventricular colistin, administered as colistin methanesulfonate (CMS), is the last resource for the treatment of central nervous system infections caused by panresistant Gram-negative bacteria. The doses and daily regimens vary considerably and are empirically chosen; the cerebrospinal fluid (CSF) pharmacokinetics of colistin after intraventricular administration of CMS has never been characterized. Nine patients (aged 18 to 73 years) were treated with intraventricular CMS (daily doses of 2.61 to 10.44 mg). Colistin concentrations were measured using a selective high-performance liquid chromatography (HPLC) assay. The population pharmacokinetics analysis was performed with the P-Pharm program. The pharmacokinetics of colistin could be best described by the one-compartment model. The estimated values (means ± standard deviations) of apparent CSF total clearance (CL/Fm, where Fm is the unknown fraction of CMS converted to colistin) and terminal half-life (t1/2λ) were 0.033 ± 0.014 liter/h and 7.8 ± 3.2 h, respectively, and the average time to the peak concentration was 3.7 ± 0.9 h. A positive correlation between CL/Fm and the amount of CSF drained (range 40 to 300 ml) was observed. When CMS was administered at doses of ≥5.22 mg/day, measured CSF concentrations of colistin were continuously above the MIC of 2 μg/ml, and measured values of trough concentration (Ctrough) ranged between 2.0 and 9.7 μg/ml. Microbiological cure was observed in 8/9 patients. Intraventricular administration of CMS at doses of ≥5.22 mg per day was appropriate in our patients, but since external CSF efflux is variable and can influence the clearance of colistin and its concentrations in CSF, the daily dose of 10 mg suggested by the Infectious Diseases Society of America may be more prudent. PMID:22687507

  11. Stability of colistin methanesulfonate in pharmaceutical products and solutions for administration to patients.

    PubMed

    Wallace, Stephanie J; Li, Jian; Rayner, Craig R; Coulthard, Kingsley; Nation, Roger L

    2008-09-01

    Colistin methanesulfonate (CMS) has the potential to hydrolyze in aqueous solution to liberate colistin, its microbiologically active and more toxic parent compound. While conversion of CMS to colistin in vivo is important for bactericidal activity, liberation of colistin during storage and/or use of pharmaceutical formulations may potentiate the toxicity of CMS. To date, there has been no information available regarding the stability of CMS in pharmaceutical preparations. Two commercial CMS formulations were investigated for stability with respect to colistin content, which was measured by a specific high-performance liquid chromatography method. Coly-Mycin M Parenteral (colistimethate lyophilized powder) was stable (<0.1% of CMS present as colistin) for at least 20 weeks at 4 degrees C and 25 degrees C at 60% relative humidity. When Coly-Mycin M was reconstituted with 2 ml of water to a CMS concentration of 200 mg/ml for injection, Coly-Mycin M was stable (<0.1% colistin formed) for at least 7 days at both 4 degrees C and 25 degrees C. When further diluted to 4 mg/ml in a glucose (5%) or saline (0.9%) infusion solution as directed, CMS hydrolyzed faster at 25 degrees C (<4% colistin formed after 48 h) than at 4 degrees C (0.3% colistin formed). The second formulation, CMS Solution for Inhalation (77.5 mg/ml), was stable at 4 degrees C and 25 degrees C for at least 12 months, as determined based on colistin content (<0.1%). This study demonstrated the concentration- and temperature-dependent hydrolysis of CMS. The information provided by this study has important implications for the formulation and clinical use of CMS products. PMID:18606838

  12. Imatinib methanesulfonate reduces hyperphosphorylation of tau following repeated peripheral exposure to lipopolysaccharide.

    PubMed

    Gardner, L E; White, J D; Eimerbrink, M J; Boehm, G W; Chumley, M J

    2016-09-01

    For years, the prevailing hypothesis for Alzheimer's Disease (AD) has proposed a mechanism by which deposition of amyloid-beta (Aβ) in the brain is independent of tau-pathologies and cognitive decline. However, despite extensive research on the disease, the mechanisms underlying the etiology of tau-pathology remain unknown. Previous research in our lab has shown that imatinib methanesulfonate (IM) blocks the peripheral production of Aβ in response to LPS, thereby preventing the buildup of Aβ in the hippocampus, and rescuing the cognitive dysfunction that normally follows. The present study aimed to examine the link between Aβ and tau following inflammation, and to expand our understanding of how IM affects AD pathology. Specifically, we hypothesized that the IM-mediated inhibition of Aβ production following inflammation would successfully protect against the hyperphosphorylation of tau (ptau). Here we show that 7days of LPS treatment in male C57BL/6J mice, which normally produces elevations in peripheral and central Aβ, also produces hyperphosphorylation of tau. However, just as pre-treatment and concurrent treatment with IM blocks Aβ production, it also blocks the phosphorylation of tau. In addition, 7days of LPS-induced inflammation and Aβ production also leads to elevated total tau protein expression. Our results may provide support for the hypothesis that enhanced expression of tau following LPS administration is a protective measure by hippocampal neurons to compensate for the loss of the microtubule-stabilizing protein due to phosphorylation. More importantly, our results support the hypothesis that blocking the production of Aβ that follows inflammation also leads to reduced tau phosphorylation, lending credence to a model in which Aβ initiates tau phosphorylation. PMID:27320209

  13. Methyl-methanesulfonate sensitivity 19 expression is associated with metastasis and chemoradiotherapy response in esophageal cancer

    PubMed Central

    Zhang, Jin-Liang; Wang, Hui-Yun; Yang, Qing; Lin, Shi-Yong; Luo, Guang-Yu; Zhang, Rong; Xu, Guo-Liang

    2015-01-01

    AIM: To investigate the clinical significance of methyl-methanesulfonate sensitivity 19 (MMS19) expression in esophageal squamous cell carcinoma (ESCC). METHODS: Between June 2008 and May 2013, specimens from 103 patients who underwent endoscopic biopsy for the diagnosis of ESCC at the endoscopy center of Sun Yat-Sen University Cancer Center were collected; 52 matched-normal esophageal squamous epithelium samples were biopsied as controls. MMS19 protein expression was measured by immunohistochemistry. Of the 103 cases of ESCC, 49 received radical surgery following neoadjuvant chemoradiotherapy consisting of concurrent radiation in a total dose of 40 Gy and two cycles of chemotherapy with vinorelbine and cisplatin. Relationships between MMS19 expression, clinicopathologic characteristics and chemoradiotherapy response were analyzed. RESULTS: The MMS19 protein could be detected in both the cytoplasm and nucleus of most specimens. High cytoplasmic expression of MMS19 was detected in 63.1% of ESCC samples, whereas high nuclear expression of MMS19 was found in 35.0%. High cytoplasmic MMS19 expression was associated with regional lymph node metastases (OR = 11.3, 95%CI: 2.3-54.7; P < 0.001) and distant metastases (OR = 13.1, 95%CI: 1.7-103.0; P = 0.002). Furthermore, high cytoplasmic MMS19 expression was associated with a response of ESCC to chemoradiotherapy (OR = 11.5, 95%CI: 3.0-44.5; P < 0.001), with a high cytoplasmic MMS19 expression rates in 79.3% and 25.0% of patients from the good chemoradiotherapy response group and poor response group, respectively. Nuclear MMS19 expression did not show any significant association with clinicopathologic characteristics or chemoradiotherapy response in ESCC. CONCLUSION: The results of our preliminary study suggest that MMS19 may be a potential new predictor of metastasis and chemoradiotherapy response in ESCC. PMID:25892874

  14. Stability of Colistin Methanesulfonate in Pharmaceutical Products and Solutions for Administration to Patients▿

    PubMed Central

    Wallace, Stephanie J.; Li, Jian; Rayner, Craig. R.; Coulthard, Kingsley; Nation, Roger L.

    2008-01-01

    Colistin methanesulfonate (CMS) has the potential to hydrolyze in aqueous solution to liberate colistin, its microbiologically active and more toxic parent compound. While conversion of CMS to colistin in vivo is important for bactericidal activity, liberation of colistin during storage and/or use of pharmaceutical formulations may potentiate the toxicity of CMS. To date, there has been no information available regarding the stability of CMS in pharmaceutical preparations. Two commercial CMS formulations were investigated for stability with respect to colistin content, which was measured by a specific high-performance liquid chromatography method. Coly-Mycin M Parenteral (colistimethate lyophilized powder) was stable (<0.1% of CMS present as colistin) for at least 20 weeks at 4°C and 25°C at 60% relative humidity. When Coly-Mycin M was reconstituted with 2 ml of water to a CMS concentration of 200 mg/ml for injection, Coly-Mycin M was stable (<0.1% colistin formed) for at least 7 days at both 4°C and 25°C. When further diluted to 4 mg/ml in a glucose (5%) or saline (0.9%) infusion solution as directed, CMS hydrolyzed faster at 25°C (<4% colistin formed after 48 h) than at 4°C (0.3% colistin formed). The second formulation, CMS Solution for Inhalation (77.5 mg/ml), was stable at 4°C and 25°C for at least 12 months, as determined based on colistin content (<0.1%). This study demonstrated the concentration- and temperature-dependent hydrolysis of CMS. The information provided by this study has important implications for the formulation and clinical use of CMS products. PMID:18606838

  15. Colistin Methanesulfonate Is an Inactive Prodrug of Colistin against Pseudomonas aeruginosa

    PubMed Central

    Bergen, Phillip J.; Li, Jian; Rayner, Craig R.; Nation, Roger L.

    2006-01-01

    There is a dearth of information on the pharmacodynamics of “colistin,” despite its increasing use as a last line of defense for treatment of infections caused by multidrug-resistant gram-negative organisms. The antimicrobial activities of colistin and colistin methanesulfonate (CMS) were investigated by studying the time-kill kinetics of each against a type culture of Pseudomonas aeruginosa in cation-adjusted Mueller-Hinton broth. The appearance of colistin from CMS spiked at 8.0 and 32 mg/liter was measured by high-performance liquid chromatography, which generated colistin concentration-time profiles. These concentration-time profiles were subsequently mimicked in other incubations, independent of CMS, by incrementally spiking colistin. When the cultures were spiked with CMS at either concentration, there was a substantial delay in the onset of the killing effect which was not evident until the concentrations of colistin generated from the hydrolysis of CMS had reached approximately 0.5 to 1 mg/liter (i.e., ∼0.5 to 1 times the MIC for colistin). The time course of the killing effect was similar when colistin was added incrementally to achieve the same colistin concentration-time course observed from the hydrolysis of CMS. Given that the killing kinetics of CMS can be accounted for by the appearance of colistin, CMS is an inactive prodrug of colistin with activity against P. aeruginosa. This is the first study to demonstrate the formation of colistin in microbiological media containing CMS and to demonstrate that CMS is an inactive prodrug of colistin. These findings have important implications for susceptibility testing involving “colistin,” in particular, for MIC measurement and for microbiological assays and pharmacokinetic and pharmacodynamic studies. PMID:16723551

  16. AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts

    PubMed Central

    Yamamoto, Ryohei; Umetsu, Makio; Yamamoto, Mizuki; Matsuyama, Satoshi; Takenaka, Shigeo; Ide, Hiroshi; Kubo, Kihei

    2015-01-01

    Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3′-OH and 5′-deoxyribose phosphate (5′-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5′-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER. PMID:25724755

  17. 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

  18. 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

  19. Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

    PubMed

    Thielmann, H W; Vosberg, H P; Reygers, U

    1975-08-15

    Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds. PMID:170107

  20. 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.

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

    PubMed

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

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene. PMID:21115006

  2. Isolation and initial characterization of a novel type of Baeyer-Villiger monooxygenase activity from a marine microorganism.

    PubMed

    Willetts, Andrew; Joint, Ian; Gilbert, Jack A; Trimble, William; Mühling, Martin

    2012-07-01

    A novel type of Baeyer-Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C-terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms. PMID:22414193

  3. Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

    PubMed Central

    Handa, Sumit; Spradling, Tyler J.; Dempsey, Daniel R.; Merkler, David J.

    2013-01-01

    Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors. PMID:22554821

  4. 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

  5. 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

  6. Role of sea ice and hemispheric circulation mode on sulphur oxidised compounds (Methanesulfonate and Sulfate) in the Artic aerosol

    NASA Astrophysics Data System (ADS)

    Becagli, Silvia; Calzolai, Giulia; Dayan, Uri; Di Biagio, Claudia; di Sarra, Alcide; Frosini, Daniele; Mazzola, Mauro; Rugi, Francesco; Severi, Mirko; Traversi, Rita; Vitale, Vito; Udisti, Roberto

    2013-04-01

    The recent decline in sea ice cover in the Arctic Ocean is expected to affect the regional radiation budget and to influence the ocean-atmosphere exchange of dimethylsulfide (DMS), thus the amount of biogenic aerosols formed from its atmospheric oxidation, such as methanesulfonate (MS-) and non-sea salt sulphate (nssSO42-). This study examines the temporal evolution of atmospheric MS- and nssSO42-, as measured in atmospheric aerosols, at Ny-Ålesund, (78.9°N, 11.9°E, Svalbard islands) and Thule (76.5°N, 68.8°W, Greenland) during three years (2010-12). Aerosol sampling was carried out using a PM10 sampler with Teflon filters, and a 12-stage impactor (SDI, Small Deposit-area Impactor) with polycarbonate filters. Analyses were performed by ion chromatography, for ion composition, and ICP-SFMS, for selected metals; both techniques are sufficiently sensitive, accurate, and reproducible to be applied to very low atmospheric load of aerosol particles, typical of remote polar regions. The evolution of MS- and nssSO4 concentrations was analysed as a function of speciation (as acidic species or ammonium salt), size distribution, and airmass pathways. This study reveals that nssSO4 is meanly associated with long range transport from anthropic sources, and presents a relative maximum in spring. Conversely, MS- arises from natural local sources and shows a peak in mid-summer. A large interannual variability is observed in MS- concentration with values in spring-summer 2010 in both the stations higher than in the other summers. In the previous winter a larger sea ice extent and larger sea ice melting surface in the following spring were observed. Arrigo et al. (2008) have observed a 22% increase in the annual primary productivity, that has been attributed to a longer phytoplankton growing season connected with the progressive decline in sea ice coverage in the Arctic over the past decade. Modeling results (Gabric et al., 2005) suggest that an increase in DMS production would

  7. 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.

  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. 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.

  11. [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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit.

    PubMed

    Sazinsky, Matthew H; Lippard, Stephen J

    2005-04-27

    The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB. PMID:15839679

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

    PubMed

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

    2014-09-17

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibriocholerae, GbpA, which is a virulence factor with no obvious role in biomass processing. PMID:25109775

  4. Alkylating agent methyl methanesulfonate (MMS) induces a wave of global protein hyperacetylation: Implications in cancer cell death

    SciTech Connect

    Lee, Min-Young; Kim, Myoung-Ae; Kim, Hyun-Ju; Bae, Yoe-Sik; Park, Joo-In; Kwak, Jong-Young; Chung, Jay H.; Yun, Jeanho . E-mail: yunj@dau.ac.kr

    2007-08-24

    Protein acetylation modification has been implicated in many cellular processes but the direct evidence for the involvement of protein acetylation in signal transduction is very limited. In the present study, we found that an alkylating agent methyl methanesulfonate (MMS) induces a robust and reversible hyperacetylation of both cytoplasmic and nuclear proteins during the early phase of the cellular response to MMS. Notably, the acetylation level upon MMS treatment was strongly correlated with the susceptibility of cancer cells, and the enhancement of MMS-induced acetylation by histone deacetylase (HDAC) inhibitors was shown to increase the cellular susceptibility. These results suggest protein acetylation is important for the cell death signal transduction pathway and indicate that the use of HDAC inhibitors for the treatment of cancer is relevant.

  5. 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

  6. 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.

  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. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Targeted deletion of kynurenine 3-monooxygenase in mice: a new tool for studying kynurenine pathway metabolism in periphery and brain.

    PubMed

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

    2013-12-20

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

  15. Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath.

    PubMed

    Stolyar, S; Costello, A M; Peeples, T L; Lidstrom, M E

    1999-05-01

    Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth. PMID:10376840

  16. 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...

  17. 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...

  18. 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...

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

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

    PubMed

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

    2013-12-01

    Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice. PMID:24371168

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Synthesis, antimicrobial and antiproliferative activity of novel silver(I) tris(pyrazolyl)methanesulfonate and 1,3,5-triaza-7-phosphadamantane complexes.

    PubMed

    Pettinari, Claudio; Marchetti, Fabio; Lupidi, Giulio; Quassinti, Luana; Bramucci, Massimo; Petrelli, Dezemona; Vitali, Luca A; da Silva, M Fátima C Guedes; Martins, Luísa M D R S; Smoleński, Piotr; Pombeiro, Armando J L

    2011-11-01

    Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)(PPh(3))] (2), [Ag(Tpms)(PCy(3))] (3), [Ag(PTA)][BF(4)] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh(3) = triphenylphosphane, PCy(3) = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane} have been synthesized and fully characterized by elemental analyses, (1)H, (13)C, and (31)P NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N(3)-facially coordinating mode, while in 2 and 5 a N(2)O-coordination is found, with the SO(3) group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA159), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA-EB system on addition of Ag complexes shows that the fluorescence quenching of DNA-EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant

  11. 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

  12. 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

  13. The Membrane-Associated Methane Monooxygenase (pMMO) and pMMO-NADH:Quinone Oxidoreductase Complex from Methylococcus capsulatus Bath

    PubMed Central

    Choi, Dong-W.; Kunz, Ryan C.; Boyd, Eric S.; Semrau, Jeremy D.; Antholine, William E.; Han, J.-I.; Zahn, James A.; Boyd, Jeffrey M.; de la Mora, Arlene M.; DiSpirito, Alan A.

    2003-01-01

    Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol · min−1 · mg of protein−1. Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 μM) to 95 μM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio. PMID:13129946

  14. The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

    PubMed

    Choi, Dong-W; Kunz, Ryan C; Boyd, Eric S; Semrau, Jeremy D; Antholine, William E; Han, J-I; Zahn, James A; Boyd, Jeffrey M; de la Mora, Arlene M; DiSpirito, Alan A

    2003-10-01

    Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio. PMID:13129946

  15. 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

  16. 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

  17. Scanning the effects of ethyl methanesulfonate on the whole genome of Lotus japonicus using second-generation sequencing analysis.

    PubMed

    Mohd-Yusoff, Nur Fatihah; Ruperao, Pradeep; Tomoyoshi, Nurain Emylia; Edwards, David; Gresshoff, Peter M; Biswas, Bandana; Batley, Jacqueline

    2015-04-01

    Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm. PMID:25660167

  18. Artichoke induces genetic toxicity and decreases ethyl methanesulfonate-related DNA damage in Chinese hamster ovary cells.

    PubMed

    Jacociunas, Laura Vicedo; de Andrade, Heloisa Helena Rodrigues; Lehmann, Mauricio; de Abreu, Bianca Regina Ribas; Ferraz, Alexandre de Barros Falcão; da Silva, Juliana; Dihl, Rafael Rodrigues

    2012-10-01

    Cynara scolymus L. (Asteraceae), popularly known as artichoke, has been widely used in herbal medicine for the treatment of hepatic diseases. The genotoxicity of C. scolymus L. leaf extract (LE) and the ability to modulate the genetic toxicity of the alkylating agent ethyl methanesulfonate (EMS) were assessed using the comet assay on Chinese hamster ovary cells. Genotoxicity was evaluated after 1- and 24-h treatments using four different LE concentrations: 0.62, 1.25, 2.5, and 5.0 mg/mL. Antigenotoxicity was assessed for pretreatment, simultaneous treatment, and post-treatment. All doses used led to a significant increase in the frequency of DNA damage, after exposure for 1 and 24 h. In the antigenotoxicity experiments, LE reduced the frequency of DNA damage induced by EMS in the simultaneous treatment only. However, the lowest dose was more protective than higher concentrations. Flavonoids and phenolic compounds are, probably, the C. scolymus constituents responsible for its genotoxic and antigenotoxic effects. PMID:22892059

  19. Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

    PubMed Central

    Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C.M.; Jansen, Jacob G.; Hogenbirk, Marc A.; de Wind, Niels; Jacobs, Heinz

    2015-01-01

    Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (PcnaK164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. PMID:25505145

  20. Variations in the methanesulfonate to sulfate molar ratio in submicrometer marine aerosol particles over the south Pacific Ocean

    NASA Technical Reports Server (NTRS)

    Bates, Timothy S.; Calhoun, Julie A.; Quinn, Patricia K.

    1992-01-01

    Seawater concentrations of dimethylsulfide (DMS) and atmospheric concentrations of DMS, sulfur dioxide, methanesulfonate (MSA), and non-sea-salt (nss) sulfate were measured over the eastern Pacific Ocean between 105 deg and 110 deg W from 20 deg N to 60 deg S during February and March 1989. Although the samples collected in the Southern Hemisphere appear to be of marine origin, no significant correlation was found between the latitudinal distributions of DMS, SO2, MSA, and nss SO4(2-). However, an inverse correlation was found between atmospheric temperature and the MSA to nss SO4(2-) molar ratio in submicrometer aerosol particles with a decrease in temperature corresponding to an increase in the molar ratio. Although this trend is consistent with laboratory results indicating the favored production of MSA at lower temperatures, it is contrary to Southern Hemisphere baseline station data. This suggests either a decrease in the supply of DMS relative to nonmarine sources of nss SO4(2-) at the baseline stations in winter or additional mechanisms that affect the relative production of MSA and nss SO4(2-).

  1. Differential action on cancer and normal tissue by adrenochrome monoaminoguanidine methanesulfonate and cytochrome C combined with radiotherapy

    SciTech Connect

    Nakatsugawa, S. ); Sugahara, T. )

    1994-06-15

    The possibility that radioprotective effects on potent natural killer (NK) cells by adrenochrome monoaminoguanidine methanesulfonate (AMM) + cytochrome C during radiotherapy (RT) for lung cancer might result in the radiosensitization of human lung cancer cells in vivo is examined. Human lung cancer xenografts in the right hind legs of KSN mice (10 weeks old) were locally irradiated with 20 Gy of X ray. AMM (10 mg/kg/day) and/or cytochrome C (CCC) (5 mg/kg/day) were given intraperitoneally immediately before or after RT, followed by daily administration for 4 days. Natural killer activities of host splenocytes were also tested with the standard [sup 51]Cr releasing assay with YAC-1 cells as target cells. In a clinical study, 65 patients with lung cancer were treated with more than 50 Gy of RT with or without combination with AMM + CCC, OK-432 or AMM + CCC + OK-432. Before and after RT, lymphocyte subsets in the peripheral blood were examined with dichromatic analysis using an Ortho Spectrum IIIFCM system and fluorescent MABs. In this study, the change in the absolute number of each subset was investigated. AMM + cytochrome C augumented NK activity in KSN nude mice, protected potent NK cells in patients with lung cancer against RT and sensitized the human lung cancer xenografts to RT. AMM + cytochrome C may have potential as a differential modulator of radiosensitivity of normal tissues and of tumors. 8 refs., 2 figs., 1 tab.

  2. Effect of mode of administration of methyl methanesulfonate and triethylenemelamine on induction of unscheduled DNA synthesis in mouse germ cells

    SciTech Connect

    Sheu, C.W.; Sega, G.A.; Owens, J.G.

    1987-01-01

    The effect of route of administration on induction of unscheduled DNA synthesis (UDS) in mouse germ cells in vivo was studied using two germ cell mutagens, methyl methanesulfonate (MMS) and triethylenemelamine (TEM). The chemicals were administered to male mice (C3Hf x 101)F/sub 1/ by IP injection or gavage using acute or 5-day subacute regimens. After completion of dosing, methyl-(/sup 3/H)thymidine ((/sup 3/H)TdR) was injected into the testes, and spermatozoa were collected 16 days later. The sperm heads were isolated, and UDS was determined by the amount of (/sup 3/H)TdR incorporated. Acute administration of MMS (2-100 mg/kg) induced a strong, dose-related UDS response. The response was slightly higher with IP injection than with gavage. Acute administration of TEM (0.05-4.0 mg/kg) by IP injection or gavage induced weak and variable responses. The study showed that gavage, as well as IP injection, can be used for the administration of test chemicals and that the subacute 5-day regimen induced a higher UDS response than the acute regimen. Furthermore, the testicular route may enhance the detection of weak UDS inducers.

  3. Self-assembly behaviour of colistin and its prodrug colistin methanesulfonate: implications for solution stability and solubilization

    PubMed Central

    Wallace, Stephanie J.; Li, Jian; Nation, Roger L.; Prankerd, Richard J.; Velkov, Tony; Boyd, Ben J.

    2010-01-01

    Colistin is an amphiphilic antibiotic that has re-emerged into clinical use due to the increasing prevalence of difficult-to-treat Gram-negative infections. The existence of self-assembling colloids in solutions of colistin and its derivative prodrug, colistin methanesulfonate (CMS) was investigated. Colistin and CMS reduced the air-water interfacial tension, and dynamic light scattering (DLS) studies showed the existence of 2.07 ± 0.3 nm aggregates above 1.5 mM for colistin, and of 1.98 ± 0.36 nm aggregates for CMS above 3.5 mM (mean ± SD). Above the respective critical micelle concentrations (CMC) the solubility of azithromycin, a hydrophobic antibiotic, increased approximately linearly with increasing surfactant concentration (5:1 mol ratio colistin:azithromycin), suggestive of hydrophobic domains within the micellar cores. Rapid conversion of CMS to colistin occurred below the CMC (60 % over 48 hr), while conversion above the CMC was less than 1 %. The formation of colistin and CMS micelles demonstrated in this study is the proposed mechanism for solubilization of azithromycin and the concentration-dependent stability of CMS. PMID:20302384

  4. Whole-genome effects of ethyl methanesulfonate-induced mutation on nine quantitative traits in outbred Drosophila melanogaster.

    PubMed

    Yang, H P; Tanikawa, A Y; Van Voorhies, W A; Silva, J C; Kondrashov, A S

    2001-03-01

    We induced mutations in Drosophila melanogaster males by treating them with 21.2 mm ethyl methanesulfonate (EMS). Nine quantitative traits (developmental time, viability, fecundity, longevity, metabolic rate, motility, body weight, and abdominal and sternopleural bristle numbers) were measured in outbred heterozygous F3 (viability) or F2 (all other traits) offspring from the treated males. The mean values of the first four traits, which are all directly related to the life history, were substantially affected by EMS mutagenesis: the developmental time increased while viability, fecundity, and longevity declined. In contrast, the mean values of the other five traits were not significantly affected. Rates of recessive X-linked lethals and of recessive mutations at several loci affecting eye color imply that our EMS treatment was equivalent to approximately 100 generations of spontaneous mutation. If so, our data imply that one generation of spontaneous mutation increases the developmental time by 0.09% at 20 degrees and by 0.04% at 25 degrees, and reduces viability under harsh conditions, fecundity, and longevity by 1.35, 0.21, and 0.08%, respectively. Comparison of flies with none, one, and two grandfathers (or greatgrandfathers, in the case of viability) treated with EMS did not reveal any significant epistasis among the induced mutations. PMID:11238409

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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...

  13. Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates.

    PubMed

    Haghbeen, Kamahldin; Wue Tan, Eng

    2003-01-01

    Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented. PMID:12479831

  14. 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.

  15. Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways.

    PubMed Central

    Van Der Werf, M J

    2000-01-01

    A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMKMO) is a monomeric enzyme of molecular mass 60 kDa. It contains 1 mol of FAD/monomer as the prosthetic group. The N-terminal amino acid sequence showed homology with many other NADPH-dependent and FAD-containing (Type 1) BVMOs. Maximal enzyme activity was measured at pH 9 and 35 degrees C. MMKMO has a broad substrate specificity, catalysing the lactonization of a large number of monocyclic monoterpene ketones and substituted cyclohexanones. The natural substrates 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone were converted stoichiometrically into 3-isopropenyl-6-oxoheptanoate (the spontaneous rearrangement product of the lactone formed by MMKMO), 4-isopropenyl-7-methyl-2-oxo-oxepanone and 7-isopropyl-4-methyl-2-oxo-oxepanone respectively. The MMKMO-catalysed conversion of iso-dihydrocarvone showed an opposite regioselectivity to that of dihydrocarvone; in this case, 6-isopropenyl-3-methyl-2-oxo-oxepanone was formed as the product. MMKMO converted all enantiomers of the natural substrates with almost equal efficiency. MMKMO is involved in the conversion of the monocyclic monoterpene ketone intermediates formed in the degradation pathways of all stereoisomers of three different monocyclic monoterpenes, i.e. limonene, (dihydro)carveol and menthol. PMID:10769172

  16. The metabolomics of (+/-)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases.

    PubMed

    Giri, Sarbani; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2007-02-15

    The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining. PMID:17123469

  17. Non-sea-salt sulfate, methanesulfonate, and nitrate aerosol concentrations and size distributions at Cape Grim, Tasmania

    NASA Astrophysics Data System (ADS)

    Andreae, Meinrat O.; Elbert, Wolfgang; Cai, Yong; Andreae, Tracey W.; Gras, John

    1999-09-01

    We collected weekly aerosol samples using high-volume impactors over a period of 20 months (1988-1990) at the Cape Grim baseline station on the northwestern coast of Tasmania, Australia. The samples were analyzed for soluble ionic constituents, including sulfate, methanesulfonate (MS-), ammonium, nitrate, and the major sea-salt ions. The sea-salt component showed only a slight seasonal variation, whereas the non-sea-salt (nss) ions all had pronounced summer maxima. Significant interannual variability was seen between the nss ion concentrations measured during the two summers investigated. Nss sulfate and MS- were present both in the fine and coarse aerosol fractions, in the latter presumably associated with sea-salt particles. During the winter period, there was more nss sulfate in the coarse fraction than in the fine fraction. These observations are consistent with an important role of liquid-phase oxidation in haze and cloud droplets for the production of nss sulfate aerosol. The seasonal behavior of the sulfur and nitrogen species at Cape Grim and their mutual correlations suggest that DMS oxidation is the dominant sulfur source during summer, while nonbiogenic sulfur sources make significant contributions to nss sulfate outside of this season. Correlations of CN and CCN concentrations with nss sulfate, MS-, and wind speed suggest that DMS oxidation and, to a lesser extent, seaspray formation contributes to CN and CCN populations. The contrast between the weak seasonality of the sea-salt component and the pronounced seasonal behavior in both sulfur species and CCN supports the central role of biogenic DMS emissions as precursors of CCN in this region, at least in the biologically productive season.

  18. Efficacy of Tricaine Methanesulfonate (MS-222) as an Anesthetic Agent for Blocking Sensory-Motor Responses in Xenopus laevis Tadpoles

    PubMed Central

    Ramlochansingh, Carlana; Branoner, Francisco; Chagnaud, Boris P.; Straka, Hans

    2014-01-01

    Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222) is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes. PMID:24984086

  19. Characterization of a starch-hydrolyzing α-amylase produced by Aspergillus niger WLB42 mutated by ethyl methanesulfonate treatment

    PubMed Central

    Wang, Shihui; Lin, Chaoyang; Liu, Yun; Shen, Zhicheng; Jeyaseelan, Jenasia; Qin, Wensheng

    2016-01-01

    Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co2+, increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase. PMID:27335681

  20. Population Pharmacokinetics of Colistin Methanesulfonate and Colistin in Critically Ill Patients with Acute Renal Failure Requiring Intermittent Hemodialysis.

    PubMed

    Jacobs, M; Grégoire, N; Mégarbane, B; Gobin, P; Balayn, D; Marchand, S; Mimoz, O; Couet, W

    2016-03-01

    Colistin is increasingly used as a last option for the treatment of severe infections due to Gram-negative bacteria in critically ill patients requiring intermittent hemodialysis (HD) for acute renal failure. Our objective was to characterize the pharmacokinetics (PK) of colistin and its prodrug colistin methanesulfonate (CMS) in this population and to suggest dosing regimen recommendations. Eight intensive care unit (ICU) patients who were under intermittent HD and who were treated by CMS (Colimycine) were included. Blood samples were collected between two consecutive HD sessions. CMS and colistin concentrations were measured by a specific chromatographic assay and were analyzed using a PK population approach (Monolix software). Monte Carlo simulations were conducted to predict the probability of target attainment (PTA). CMS nonrenal clearance was increased in ICU-HD patients. Compared with that of ICU patients included in the same clinical trial but with preserved renal function, colistin exposure was increased by 3-fold in ICU-HD patients. This is probably because a greater fraction of the CMS converted into colistin. To maintain colistin plasma concentrations high enough (>3 mg/liter) for high PTA values (area under the concentration-time curve for the free, unbound fraction of a drug [fAUC]/MIC of >10 and fAUC/MIC of >50 for systemic and lung infections, respectively), at least for MICs lower than 1.5 mg/liter (nonpulmonary infection) or 0.5 mg/liter (pulmonary infection), the dosing regimen of CMS should be 1.5 million international units (MIU) twice daily on non-HD days. HD should be conducted at the end of a dosing interval, and a supplemental dose of 1.5 MIU should be administered after the HD session (i.e., total of 4.5 MIU for HD days). This study has confirmed and complemented previously published data and suggests an a priori clear and easy to follow dosing strategy for CMS in ICU-HD patients. PMID:26729492

  1. Characterization of a starch-hydrolyzing α-amylase produced by Aspergillus niger WLB42 mutated by ethyl methanesulfonate treatment.

    PubMed

    Wang, Shihui; Lin, Chaoyang; Liu, Yun; Shen, Zhicheng; Jeyaseelan, Jenasia; Qin, Wensheng

    2016-01-01

    Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co(2+), increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase. PMID:27335681

  2. Population Pharmacokinetics of Colistin Methanesulfonate in Rats: Achieving Sustained Lung Concentrations of Colistin for Targeting Respiratory Infections

    PubMed Central

    W. S. Yapa, Shalini; Li, Jian; Porter, Christopher J. H.; Nation, Roger L.

    2013-01-01

    Colistin methanesulfonate (CMS), the inactive prodrug of colistin, is administered by inhalation for the management of respiratory infections. However, limited pharmacokinetic data are available for CMS and colistin following pulmonary delivery. This study investigates the pharmacokinetics of CMS and colistin following intravenous (i.v.) and intratracheal (i.t.) administration in rats and determines the targeting advantage after direct delivery into the lungs. In addition to plasma, bronchoalveolar lavage (BAL) fluid was collected to quantify drug concentrations in lung epithelial lining fluid (ELF). The resulting data were analyzed using a population modeling approach in S-ADAPT. A three-compartment model described the disposition of both compounds in plasma following i.v. administration. The estimated mean clearance from the central compartment was 0.122 liters/h for CMS and 0.0657 liters/h for colistin. Conversion of CMS to colistin from all three compartments was required to fit the plasma data. The fraction of the i.v. dose converted to colistin in the systemic circulation was 0.0255. Two BAL fluid compartments were required to reflect drug kinetics in the ELF after i.t. dosing. A slow conversion of CMS (mean conversion time [MCTCMS] = 3.48 h) in the lungs contributed to high and sustained concentrations of colistin in ELF. The fraction of the CMS dose converted to colistin in ELF (fm,ELF = 0.226) was higher than the corresponding fractional conversion in plasma after i.v. administration. In conclusion, pulmonary administration of CMS achieves high and sustained exposures of colistin in lungs for targeting respiratory infections. PMID:23917323

  3. Stability of Colistin and Colistin Methanesulfonate in Aqueous Media and Plasma as Determined by High-Performance Liquid Chromatography

    PubMed Central

    Li, Jian; Milne, Robert W.; Nation, Roger L.; Turnidge, John D.; Coulthard, Kingsley

    2003-01-01

    The stabilities of colistin and colistin methanesulfonate (CMS) in different aqueous media were studied by specific high-performance liquid chromatography (HPLC) methods. Colistin was stable in water at 4 and 37°C for up to 60 days and 120 h, respectively. However, degradation was observed when colistin was stored in isotonic phosphate buffer (0.067 M, pH 7.4) and human plasma at 37°C. The stability of CMS from three different sources in water was explored by strong-anion-exchange (SAX) HPLC for CMS and by measuring the concentrations of colistin formed from the hydrolysis of CMS. The peaks of CMS in SAX HPLC disappeared almost completely after 12 h at 37°C, but appeared to remain intact for up to 2 days at 4°C. Over the same period, there was no formation of colistin at 4°C. In water, phosphate buffer, and plasma, there was rapid formation of colistin within 24 to 48 h at 37°C from the three sources of CMS. The hydrolysis products were assumed to be a complex mixture of many different sulfomethyl derivatives, including colistin. The stability of a fourth source of CMS in Mueller-Hinton broth examined during 30 min at 37°C revealed no formation of colistin. Along with previous microbiological studies, this suggested that different sulfomethyl CMSs possess intrinsic antibacterial activity. These results will be helpful for understanding the pharmacokinetics and pharmacodynamics of colistin and CMS in humans and animals. PMID:12654671

  4. Pulmonary and Systemic Pharmacokinetics of Inhaled and Intravenous Colistin Methanesulfonate in Cystic Fibrosis Patients: Targeting Advantage of Inhalational Administration

    PubMed Central

    W. S. Yapa, Shalini; Li, Jian; Patel, Kashyap; Wilson, John W.; Dooley, Michael J.; George, Johnson; Clark, Denise; Poole, Susan; Williams, Elyssa; Porter, Christopher J. H.

    2014-01-01

    The purpose of this study was to define the pulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and formed colistin following intravenous (i.v.) and inhaled administration in cystic fibrosis (CF) patients. Six CF subjects were administered nebulized CMS doses of 2 and 4 million IU and an i.v. CMS infusion of 150 mg of colistin base activity. Blood plasma, sputum, and urine samples were collected for 12 to 24 h postdose. To assess the tolerability of the drug, lung function tests, blood serum creatinine concentrations, and adverse effect reports were recorded. All doses were well tolerated in the subjects. The pharmacokinetic parameters for CMS following i.v. delivery were consistent with previously reported values. Sputum concentrations of formed colistin were maintained at <1.0 mg/liter for 12 h postdose. Nebulization of CMS resulted in relatively high sputum concentrations of CMS and formed colistin compared to those resulting from i.v. administration. The systemic availability of CMS was low following nebulization of 2 and 4 million IU (7.93% ± 4.26% and 5.37% ± 1.36%, respectively), and the plasma colistin concentrations were below the limit of quantification. Less than 2 to 3% of the nebulized CMS dose was recovered in the urine samples in 24 h. The therapeutic availability and drug targeting index for CMS and colistin following inhalation compared to i.v. delivery were significantly greater than 1. Inhalation of CMS is an effective means of targeting CMS and formed colistin for delivery to the lungs, as high lung exposure and minimal systemic exposure were achieved in CF subjects. PMID:24550334

  5. 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

  6. Differential Reactivity between Two Copper Sites in Peptidylglycine r-Hydroxylating Monooxygenase

    SciTech Connect

    E Chufan; S Prigge; X Siebert; B Eipper; R Mains; L Amzel

    2011-12-31

    Peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the C{alpha} of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu{sub M} and Cu{sub H}) separated by {approx}11 {angstrom}. In the resting state of the enzyme, Cu{sub M} is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu{sub H} has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu{sub M} is identified as the metal involved in catalysis, while Cu{sub H} is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO{sub 2}{sup -}), azide (N{sub 3}{sup -}), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO{sub 2}, shows that nitrite anion coordinates Cu{sub M} in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu{sub H}, despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu{sub M} but not Cu{sub H} in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN{sub 3} and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu{sub H} is also observed in the reduced form of the enzyme: CO binds Cu{sub M} but not Cu{sub H} in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5

  7. Contribution of ethyl methanesulfonate vapors to the yield of mutations detected in Drosophila melanogaster when the adult feeding technique is used

    SciTech Connect

    Munoz, E.R.

    1987-01-01

    Ethyl methanesulfonate (EMS) is an alkylating agent widely used in mutation research. In experiments with adult Drosophila melanogaster, EMS is either injected or fed to the flies using different feeding methods that essentially consist of placing the flies in bottles or vials with a piece of tissue paper moistened with a sucrose solution containing the desired concentration of EMS. To determine the extent to which vapors contribute to the mutagenic effect detected in Drosophila when the feeding technique is used, 7-day-old wild-type Samarkand males were fed EMS or were exposed only to its vapors.

  8. Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium.

    PubMed

    Wu, Miao; Beckham, Gregg T; Larsson, Anna M; Ishida, Takuya; Kim, Seonah; Payne, Christina M; Himmel, Michael E; Crowley, Michael F; Horn, Svein J; Westereng, Bjørge; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Eijsink, Vincent G H; Sandgren, Mats

    2013-05-01

    Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. PMID:23525113

  9. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s.

    PubMed

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E; Nelson, David R; Tuszynski, Jack A; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-01-01

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria -42; fungi -19; plant -28; animal -22; plant and animal -1 and common P450 family -1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study's results offer new understanding of the dynamic structural nature of P450s. PMID:27616185

  10. Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species

    PubMed Central

    Sales, Christopher M.; Grostern, Ariel; Parales, Juanito V.; Parales, Rebecca E.

    2013-01-01

    The bacterium Pseudonocardia dioxanivorans CB1190 grows on the cyclic ethers 1,4-dioxane (dioxane) and tetrahydrofuran (THF) as sole carbon and energy sources. Prior transcriptional studies indicated that an annotated THF monooxygenase (THF MO) gene cluster, thmADBC, located on a plasmid in CB1190 is upregulated during growth on dioxane. In this work, transcriptional analysis demonstrates that upregulation of thmADBC occurs during growth on the dioxane metabolite β-hydroxyethoxyacetic acid (HEAA) and on THF. Comparison of the transcriptomes of CB1190 grown on THF and succinate (an intermediate of THF degradation) permitted the identification of other genes involved in THF metabolism. Dioxane and THF oxidation activity of the THF MO was verified in Rhodococcus jostii RHA1 cells heterologously expressing the CB1190 thmADBC gene cluster. Interestingly, these thmADBC expression clones accumulated HEAA as a dead-end product of dioxane transformation, indicating that despite its genes being transcriptionally upregulated during growth on HEAA, the THF MO enzyme is not responsible for degradation of HEAA in CB1190. Similar activities were also observed in RHA1 cells heterologously expressing the thmADBC gene cluster from Pseudonocardia tetrahydrofuranoxydans K1. PMID:24096414

  11. Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

    PubMed

    Abdelwahab, Heba; Robinson, Reeder; Rodriguez, Pedro; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

    2016-09-15

    l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding. PMID:27503802

  12. Isolation of Homogeneous Polysaccharide Monooxygenases from Fungal Sources and Investigation of Their Synergism with Cellulases when Acting on Cellulose.

    PubMed

    Bulakhov, A G; Gusakov, A V; Chekushina, A V; Satrutdinov, A D; Koshelev, A V; Matys, V Yu; Sinitsyn, A P

    2016-05-01

    Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes - Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila - were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 µM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction. PMID:27297903

  13. Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium*

    PubMed Central

    Wu, Miao; Beckham, Gregg T.; Larsson, Anna M.; Ishida, Takuya; Kim, Seonah; Payne, Christina M.; Himmel, Michael E.; Crowley, Michael F.; Horn, Svein J.; Westereng, Bjørge; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Eijsink, Vincent G. H.; Sandgren, Mats

    2013-01-01

    Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. PMID:23525113

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100

    PubMed Central

    Gisi, Michelle R.; Xun, Luying

    2003-01-01

    Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH2)-utilizing monooxygenase, and FADH2 was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH2 and retarded its rapid oxidation by O2. A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH2-utilizing enzymes, which have homologues in the domains Bacteria and Archaea. PMID:12700257

  6. Colistin Population Pharmacokinetics after Application of a Loading Dose of 9 MU Colistin Methanesulfonate in Critically Ill Patients

    PubMed Central

    Friberg, Lena E.; Pontikis, Konstantinos; Ioannidis, Konstantinos; Tsagkari, Vasiliki; Galani, Lamprini; Kostakou, Eirini; Baziaka, Fotini; Paskalis, Charalambos; Koutsoukou, Antonia; Giamarellou, Helen

    2015-01-01

    Colistin has been revived, in the era of extensively drug-resistant (XDR) Gram-negative infections, as the last-resort treatment in critically ill patients. Recent studies focusing on the optimal dosing strategy of colistin have demonstrated the necessity of a loading dose at treatment initiation (D. Plachouras, M. Karvanen, L. E. Friberg, E. Papadomichelakis, A. Antoniadou, I. Tsangaris, I. Karaiskos, G. Poulakou, F. Kontopidou, A. Armaganidis, O. Cars, and H. Giamarellou, Antimicrob Agents Chemother 53:3430–3436, 2009, http://dx.doi.org/10.1128/AAC.01361-08; A. F. Mohamed, I. Karaiskos, D. Plachouras, M. Karvanen, K. Pontikis, B. Jansson, E. Papadomichelakis, A. Antoniadou, H. Giamarellou, A. Armaganidis, O. Cars, and L. E. Friberg, Antimicrob Agents Chemother 56:4241– 4249, 2012, http://dx.doi.org/10.1128/AAC.06426-11; S. M. Garonzik, J. Li, V. Thamlikitkul, D. L. Paterson, S. Shoham, J. Jacob, F. P. Silveira, A. Forrest, and R. L. Nation, Antimicrob Agents Chemother 55:3284–3294, 2011, http://dx.doi.org/10.1128/AAC.01733-10). In 19 critically ill patients with suspected or microbiologically documented infections caused by XDR Gram-negative strains, a loading dose of 9 MU colistin methanesulfonate (CMS) (∼270 mg colistin base activity) was administered with a maintenance dose of 4.5 MU every 12 h, commenced after 24 h. Patients on renal replacement were excluded. CMS infusion was given over 30 min or 1 h. Repeated blood sampling was performed after the loading dose and after the 5th or 6th dose. Colistin concentrations and measured CMS, determined after hydrolization to colistin and including the partially sulfomethylated derivatives, were determined with a liquid chromatography-tandem mass spectrometry assay. Population pharmacokinetic analysis was conducted in NONMEM with the new data combined with data from previous studies. Measured colistimethate concentrations were described by 4 compartments for distribution and removal of sulfomethyl groups

  7. Colistin Population Pharmacokinetics after Application of a Loading Dose of 9 MU Colistin Methanesulfonate in Critically Ill Patients.

    PubMed

    Karaiskos, Ilias; Friberg, Lena E; Pontikis, Konstantinos; Ioannidis, Konstantinos; Tsagkari, Vasiliki; Galani, Lamprini; Kostakou, Eirini; Baziaka, Fotini; Paskalis, Charalambos; Koutsoukou, Antonia; Giamarellou, Helen

    2015-12-01

    Colistin has been revived, in the era of extensively drug-resistant (XDR) Gram-negative infections, as the last-resort treatment in critically ill patients. Recent studies focusing on the optimal dosing strategy of colistin have demonstrated the necessity of a loading dose at treatment initiation (D. Plachouras, M. Karvanen, L. E. Friberg, E. Papadomichelakis, A. Antoniadou, I. Tsangaris, I. Karaiskos, G. Poulakou, F. Kontopidou, A. Armaganidis, O. Cars, and H. Giamarellou, Antimicrob Agents Chemother 53:3430-3436, 2009, http://dx.doi.org/10.1128/AAC.01361-08; A. F. Mohamed, I. Karaiskos, D. Plachouras, M. Karvanen, K. Pontikis, B. Jansson, E. Papadomichelakis, A. Antoniadou, H. Giamarellou, A. Armaganidis, O. Cars, and L. E. Friberg, Antimicrob Agents Chemother 56:4241- 4249, 2012, http://dx.doi.org/10.1128/AAC.06426-11; S. M. Garonzik, J. Li, V. Thamlikitkul, D. L. Paterson, S. Shoham, J. Jacob, F. P. Silveira, A. Forrest, and R. L. Nation, Antimicrob Agents Chemother 55:3284-3294, 2011, http://dx.doi.org/10.1128/AAC.01733-10). In 19 critically ill patients with suspected or microbiologically documented infections caused by XDR Gram-negative strains, a loading dose of 9 MU colistin methanesulfonate (CMS) (∼ 270 mg colistin base activity) was administered with a maintenance dose of 4.5 MU every 12 h, commenced after 24 h. Patients on renal replacement were excluded. CMS infusion was given over 30 min or 1 h. Repeated blood sampling was performed after the loading dose and after the 5th or 6th dose. Colistin concentrations and measured CMS, determined after hydrolization to colistin and including the partially sulfomethylated derivatives, were determined with a liquid chromatography-tandem mass spectrometry assay. Population pharmacokinetic analysis was conducted in NONMEM with the new data combined with data from previous studies. Measured colistimethate concentrations were described by 4 compartments for distribution and removal of sulfomethyl groups, while

  8. Sulfur and Hydrogen Isotope Anomalies in Meteorite Sulfonic Acids

    NASA Technical Reports Server (NTRS)

    Cooper, George W.; Thiemens, Mark H.; Jackson, Teresa L.; Chang, Sherwood

    1997-01-01

    Intramolecular carbon, hydrogen, and sulfur isotope ratios were measured on a homologous series of organic sulfonic acids discovered in the Murchison meteorite. Mass-independent sulfur isotope fractionations were observed along with high deuterium/hydrogen ratios. The deuterium enrichments indicate formation of the hydrocarbon portion of these compounds in a low-temperature environment that is consistent with that of interstellar clouds. Sulfur-33 enrichments observed in methanesulfonic acid could have resulted from gas-phase ultraviolet irradiation of a precursor, carbon disulfide. The source of the sulfonic acid precursors may have been the reactive interstellar molecule carbon monosulfide.

  9. Allium cepa anaphase-telophase root tip chromosome aberration assay on N-methyl-N-nitrosourea, maleic hydrazide, sodium azide, and ethyl methanesulfonate.

    PubMed

    Rank, J; Nielsen, M H

    1997-04-24

    The Allium anaphase-telophase assay was used to show genotoxicity of N-methyl-N-nitrosourea (MNU), maleic hydrazide (MH), sodium azide (NaN3) and ethyl methanesulfonate (EMS). All agents induced chromosome aberrations at statistically significant levels. The rank of the lowest doses with positive effect was as follows: NaN3 0.3 mg/l < MH 1 mg/l < MNU 41 mg/l < EMS 100 mg/l. The results were compared with results from other plant assays (Arabidopsis, Vicia, Tradescantia) and for MH and MNU the values were found to be within the same range, whereas the results in the Allium test for NaN3 and EMS were in a lower range than that found for the other plant assays. EMS and MMS (methyl methanesulfonate), two chemicals used as positive controls in mutagenicity testing, were compared in the Allium test, and MMS was found to be about ten times more potent in inducing chromosome aberrations than EMS. Recording of micronuclei in interphase cells showed that this endpoint does not give more information of clastogenicity than recording of chromosome aberrations in anaphase-telophase cells. PMID:9150760

  10. Oxorhenium complexes bearing the water-soluble tris(pyrazol-1-yl)methanesulfonate, 1,3,5-triaza-7-phosphaadamantane, or related ligands, as catalysts for Baeyer-Villiger oxidation of ketones.

    PubMed

    Martins, Luísa M D R S; Alegria, Elisabete C B A; Smoleński, Piotr; Kuznetsov, Maxim L; Pombeiro, Armando J L

    2013-04-15

    New rhenium(VII or III) complexes [ReO3(PTA)2][ReO4] (1) (PTA = 1,3,5-triaza-7-phosphaadamantane), [ReO3(mPTA)][ReO4]I (2) (mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation), [ReO3(HMT)2][ReO4] (3) (HMT = hexamethylenetetramine), [ReO3(η(2)-Tpm)(PTA)][ReO4] (4) [Tpm = hydrotris(pyrazol-1-yl)methane, HC(pz)3, pz = pyrazolyl], [ReO3(Hpz)(HMT)][ReO4] (5) (Hpz = pyrazole), [ReO(Tpms)(HMT)] (6) [Tpms = tris(pyrazol-1-yl)methanesulfonate, O3SC(pz)3(-)] and [ReCl2{N2C(O)Ph}(PTA)3] (7) have been prepared from the Re(VII) oxide Re2O7 (1-6) or, in the case of 7, by ligand exchange from the benzoyldiazenido complex [ReCl2{N2C(O)Ph}(Hpz)(PPh3)2], and characterized by IR and NMR spectroscopies, elemental analysis and electrochemical properties. Theoretical calculations at the density functional theory (DFT) level of theory indicated that the coordination of PTA to both Re(III) and Re(VII) centers by the P atom is preferable compared to the coordination by the N atom. This is interpreted in terms of the Re-PTA bond energy and hard-soft acid-base theory. The oxo-rhenium complexes 1-6 act as selective catalysts for the Baeyer-Villiger oxidation of cyclic and linear ketones (e.g., 2-methylcyclohexanone, 2-methylcyclopentanone, cyclohexanone, cyclopentanone, cyclobutanone, and 3,3-dimethyl-2-butanone or pinacolone) to the corresponding lactones or esters, in the presence of aqueous H2O2. The effects of a variety of factors are studied toward the optimization of the process. PMID:23534942

  11. Regiochemical variations in reactions of methylcubane with tert-butoxyl radical, cytochrome P-450 enzymes, and a methane monooxygenase system

    SciTech Connect

    Choi, S.Y.; Hollenberg, P.F.; Newcomb, M.; Putt, D.A.; Eaton, P.E.; Upadhyaya, S.P.; Xiong, Y.; Liu, K.E.; Lippard, S.J.

    1996-07-17

    Reactions of methylcubane (1) with the tert-butoxyl radical (t-BuO{sup .}), with cytochrome P-450 enzymes, and with a methane monooxygenase (MMO) system have been studied. 2-Methylcubanecarboxylic acid (9b) is a new compound prepared from cubanecarboxylic acid. The key synthetic reactions were (1) metalation and subsequent iodination of the 2-position of (diisopropylcarbamoyl)cubane to effect the initial functionalization, (2) lithium-for-iodine exchange and methylation followed by reduction to give 2-methyl-l-[(diisopropylamino)methyl]-cubane, and (3) dimethyldioxirane oxidation of this amine to give 9b. Reaction of 1 with t-BuO{sup .} in the presence of 2,2,5,5-tetramethylisoindole-N-oxyl radical (TMIO{sup .}) at 40-55{degree}C gave mainly cube-substituted products in confirmation of the report that hydrogen atom abstraction by the electrophilic alkoxyl radical at low temperature occurs at the cubyl C-H positions. In a competition experiment at 42{degree}C, methylcubane was at least 3.5 times more reactive toward t-BuO{sup .} than cyclohexane, indicating that the cubyl positions in 1 are >= 40 times more reactive than the methyl positions in 1 (per hydrogen) toward the alkoxyl radical. Oxidation of 1 by enzymes gave alcohol products that were converted to their acetate derivatives for identification and quantitation. Microsomal cytochrome P-450 enzymes from rat and the rat purified P-450 isozyme CYP2B1 hydroxylated 1 at all positions, whereas the reconstituted MMO system from Methylococcus capsulatus (Bath) hydroxylated l only at the methyl position. 78 refs., 1 tab.

  12. 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

  13. 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

  14. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase

    SciTech Connect

    Baldwin, J.; Voegtli, W.C.; Khidekel, N.; Moënne-Loccoz, P.; Krebs, C.; Ley, B.A.; Huynh, B.H.; Loehr, T.M.; Rosenzweig, A.C.; Bollinger, Jr., J.M.

    2010-03-05

    The outcome of O{sub 2} activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine){sub 2}-(carboxylate){sub 4} ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a {mu}-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H{sub peroxo}) in O{sub 2} activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the 'extra' electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the {mu}-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 and which converts very slowly (t{sub 1/2} {approx} 7 h) upon incubation at 0 C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone {epsilon}-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with {sup 16}O{sub 2} or {sub 18}O{sub 2} show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O{sub 2}. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Moessbauer and EXAFS characterization of the brown

  15. 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

  16. 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...

  17. 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

  18. 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

  19. 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

  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. 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

  4. Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases.

    PubMed

    Forsberg, Zarah; Røhr, Asmund Kjendseth; Mekasha, Sophanit; Andersson, K Kristoffer; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav; Sørlie, Morten

    2014-03-18

    Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality. PMID:24559135

  5. Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions.

    PubMed

    Rosenzweig, A C; Brandstetter, H; Whittington, D A; Nordlund, P; Lippard, S J; Frederick, C A

    1997-10-01

    The crystal structure of the nonheme iron-containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 A resolution. The enzyme is composed of two copies each of three subunits (alpha 2 beta 2 gamma 2), and all three subunits are almost completely alpha-helical, with the exception of two beta hairpin structures in the alpha subunit. The active site of each alpha subunit contains one dinuclear iron center, housed in a four-helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4 degrees C, a bridging acetate ion, which is replaced at -160 degrees C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural differences identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position. PMID:9329079

  6. Structural characterization by EXAFS spectroscopy of the binuclear iron center in protein A of methane monooxygenase from methylococcus capsulatus (bath)

    SciTech Connect

    Ericson, A.; Hedman, B.; Hodgson, K.O.; Green, J.; Dalton, H.; Bentsen, J.G.; Beer, R.H.; Lippard, S.J.

    1988-03-30

    Soluble methane monooxygenase (MMO) from Methylococcus capsulatus (Bath) activates dioxygen for incorporation into a remarkable variety of substrates including methane, which is required for bacterial growth (eq 1). MMO is a three component CH/sub 4/ + NADH + H/sup +/ + O/sub 2/ /sup MMO/ ..-->.. CH/sub 3/OH + NAD/sup +/ + H/sub 2/O (1) enzyme. Protein A (M/sub r/ 210,000), believed to be the oxygenase component, contains two iron atoms. Protein B (M/sub r/ 15,700) serves a regulatory function and lacks prosthetic groups, while protein C, the reductase component of the enzyme, is an iron-sulfur flavoprotein (M/sub r/ 42,000) responsible for electron transfer from NADH to protein A. Recently, a binuclear iron center was postulated to occur in protein A based on the finding that one-electron reduction gives rise to electron spin resonance (ESR) signals (g 1.95, 1.88, 1.78) very similar to those observed for the binuclear mixed-valence Fe/sub 2/(III,II) centers in semimet hemerythrin (Hr) and purple acid phosphatase (PAP). In conjunction with model studies, extended X-ray absorption fine structure (EXAFS) spectroscopy has proven to be a powerful method for identifying bridged binuclear iron centers in Hr, ribonucleotide reductase (RR), and PAP. Here the authors report iron K-edge EXAFS results on semireduced protein A of MMO which support the occurrence of a binuclear iron center (Fe-Fe distance, 3.41 A), with no short ..mu..-oxo bridge.

  7. CYP709B3, a cytochrome P450 monooxygenase gene involved in salt tolerance in Arabidopsis thaliana

    PubMed Central

    2013-01-01

    Background Within the Arabidopsis genome, there are 272 cytochrome P450 monooxygenase (P450) genes. However, the biological functions of the majority of these P450s remain unknown. The CYP709B family of P450s includes three gene members, CYP709B1, CYP709B2 and CYP709B3, which have high amino acid sequence similarity and lack reports elucidating biological functions. Results We identified T-DNA insertion-based null mutants of the CYP709B subfamily of genes. No obvious morphological phenotypes were exhibited under normal growth conditions. When the responses to ABA and salt stress were studied in these mutants, only the cyp709b3 mutant showed sensitivity to ABA and salt during germination. Under moderate salt treatment (150 mM NaCl), cyp709b3 showed a higher percentage of damaged seedlings, indicating a lower tolerance to salt stress. CYP709B3 was highly expressed in all analyzed tissues and especially high in seedlings and leaves. In contrast, CYP709B1 and CYP709B2 were highly expressed in siliques, but were at very low levels in other tissues. Under salt stress condition, CYP709B3 gene expression was induced after 24 hr and remained at high expression level. Expression of the wild type CYP709B3 gene in the cyp709b3 mutant fully complemented the salt intolerant phenotype. Furthermore, metabolite profiling analysis revealed some differences between wild type and cyp709b3 mutant plants, supporting the salt intolerance phenotype of the cyp709b3 mutant. Conclusions These results suggest that CYP709B3 plays a role in ABA and salt stress response and provides evidence to support the functions of cytochrome P450 enzymes in plant stress response. PMID:24164720

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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...

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

    PubMed

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

    2016-08-01

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

  7. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations.

    PubMed

    Rotthauwe, J H; Witzel, K P; Liesack, W

    1997-12-01

    The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the beta subclass of the Proteobacteria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA). The PCR yielded a specific 491-bp fragment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of methane-oxidizing bacteria. The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments. The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries. No false positives were detected in a set of 47 randomly selected clone sequences that were analyzed further. The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira. The comparative analysis identified members of both of these genera in activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots. Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations are the main colonizers of the rhizosphere of rice. Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with the Nitrosospira cluster but could not be assigned to any of the known pure cultures. Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine

  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. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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...

  19. The Preference for Error-Free or Error-Prone Postreplication Repair in Saccharomyces cerevisiae Exposed to Low-Dose Methyl Methanesulfonate Is Cell Cycle Dependent

    PubMed Central

    Huang, Dongqing; Piening, Brian D.

    2013-01-01

    Cells employ error-free or error-prone postreplication repair (PRR) processes to tolerate DNA damage. Here, we present a genome-wide screen for sensitivity to 0.001% methyl methanesulfonate (MMS). This relatively low dose is of particular interest because wild-type cells exhibit no discernible phenotypes in response to treatment, yet PRR mutants are unique among repair mutants in their exquisite sensitivity to 0.001% MMS; thus, low-dose MMS treatment provides a distinctive opportunity to study postreplication repair processes. We show that upon exposure to low-dose MMS, a PRR-defective rad18Δ mutant stalls into a lengthy G2 arrest associated with the accumulation of single-stranded DNA (ssDNA) gaps. Consistent with previous results following UV-induced damage, reactivation of Rad18, even after prolonged G2 arrest, restores viability and genome integrity. We further show that PRR pathway preference in 0.001% MMS depends on timing and context; cells preferentially employ the error-free pathway in S phase and do not require MEC1-dependent checkpoint activation for survival. However, when PRR is restricted to the G2 phase, cells utilize REV3-dependent translesion synthesis, which requires a MEC1-dependent delay and results in significant hypermutability. PMID:23382077

  20. Delineating the antigenotoxic and anticytotoxic potentials of 4-methylimidazole against ethyl methanesulfonate toxicity in bone marrow cell of swiss albino mice.

    PubMed

    Norizadeh Tazehkand, M; Topaktas, M; Yilmaz, M B; Hajipour, O; Valipour, E

    2016-01-01

    4-Methylimidazole (4-MEI) is mostly used in beverages and coloring food, dark beers and common brands of cola drinks, which may contain more than 100 μg of this compound per 12-ounce serving. This study was aimed to investigate the antigenotoxic and anticytotoxic effects of 4-MEI (100, 130 and 160 mg/kg) against ethyl methanesulfonate (240 mg/kg) using chromosome aberrations (CAs) and Mitotic index (MI) tests in bone marrow cells of Swiss Albino Mice at 12 h and 24 h treatment periods. So, the t-test was used for the statistical analysis.In this research, 4-MEI at all concentrations for 12 h treatment period reduced chromosomal aberrations and at 130 and 160 mg/kg concentrations for 24 h treatment period increased chromosomal aberrations induced by EMS (240 mg/kg), but th ese reductions and increases were not significant. Also, intraperitoneal injection of 4-MEI at doses of 100, 130 and 160 mg/kg combined with EMS (240 mg/kg) showed that the mitotic index was decreased at 100 and 130 mg/kg for 12h and 130 mg/kg for 24 h treatment periods, when compared to positive sample (EMS), but did not show any statistically difference from the EMS treated group. It can be concluded that 4-MEI might not be antigenotoxic and protective effects in bone marrow cells of Swiss Albino Mice, because 4-MEI could not reduce the chromosomal aberrations induced by EMS. PMID:27215965

  1. Sulfonate salts of the therapeutic agent dapsone: 4-[(4-aminophenyl)sulfonyl]anilinium benzenesulfonate monohydrate and 4-[(4-aminophenyl)sulfonyl]anilinium methanesulfonate monohydrate.

    PubMed

    Gaytán-Barrientos, Nancy Sarahy; Morales-Morales, David; Herrera-Ruiz, Dea; Reyes-Martínez, Reyna; Rivera-Islas, Jesús

    2016-04-01

    Dapsone, formerly used to treat leprosy, now has wider therapeutic applications. As is the case for many therapeutic agents, low aqueous solubility and high toxicity are the main problems associated with its use. Derivatization of its amino groups has been widely explored but shows no significant therapeutic improvements. Cocrystals have been prepared to understand not only its structural properties, but also its solubility and dissolution rate. Few salts of dapsone have been described. The title salts, C12H13N2O2S(+)·C6H5O3S(-)·H2O and C12H13N2O2S(+)·CH3SO3(-)·H2O, crystallize as hydrates and both compounds exhibit the same space group (monoclinic, P21/n). The asymmetric unit of each salt consists of a 4-[(4-aminophenyl)sulfonyl]anilinium monocation, the corresponding sulfonate anion and a water molecule. The cation, anion and water molecule form hydrogen-bonded networks through N-H...O=S, N-H...Owater and Owater-H...O=S hydrogen bonds. For both salts, the water molecules interact with one sulfonate anion and two anilinium cations. The benzenesulfonate salt forms a two-dimensional network, while the hydrogen bonding within the methanesulfonate salt results in a three-dimensional network. PMID:27045177

  2. Comparison of Intrapulmonary and Systemic Pharmacokinetics of Colistin Methanesulfonate (CMS) and Colistin after Aerosol Delivery and Intravenous Administration of CMS in Critically Ill Patients

    PubMed Central

    Boisson, Matthieu; Jacobs, Matthieu; Grégoire, Nicolas; Gobin, Patrice; Marchand, Sandrine; Mimoz, Olivier

    2014-01-01

    Colistin is an old antibiotic that has recently gained a considerable renewal of interest for the treatment of pulmonary infections due to multidrug-resistant Gram-negative bacteria. Nebulization seems to be a promising form of administration, but colistin is administered as an inactive prodrug, colistin methanesulfonate (CMS); however, differences between the intrapulmonary concentrations of the active moiety as a function of the route of administration in critically ill patients have not been precisely documented. In this study, CMS and colistin concentrations were measured on two separate occasions within the plasma and epithelial lining fluid (ELF) of critically ill patients (n = 12) who had received 2 million international units (MIU) of CMS by aerosol delivery and then intravenous administration. The pharmacokinetic analysis was conducted using a population approach and completed by pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulations. The ELF colistin concentrations varied considerably (9.53 to 1,137 mg/liter), but they were much higher than those in plasma (0.15 to 0.73 mg/liter) after aerosol delivery but not after intravenous administration of CMS. Following CMS aerosol delivery, typically, 9% of the CMS dose reached the ELF, and only 1.4% was presystemically converted into colistin. PK-PD analysis concluded that there was much higher antimicrobial efficacy after CMS aerosol delivery than after intravenous administration. These new data seem to support the use of aerosol delivery of CMS for the treatment of pulmonary infections in critical care patients. PMID:25267660

  3. Comparison of intrapulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and colistin after aerosol delivery and intravenous administration of CMS in critically ill patients.

    PubMed

    Boisson, Matthieu; Jacobs, Matthieu; Grégoire, Nicolas; Gobin, Patrice; Marchand, Sandrine; Couet, William; Mimoz, Olivier

    2014-12-01

    Colistin is an old antibiotic that has recently gained a considerable renewal of interest for the treatment of pulmonary infections due to multidrug-resistant Gram-negative bacteria. Nebulization seems to be a promising form of administration, but colistin is administered as an inactive prodrug, colistin methanesulfonate (CMS); however, differences between the intrapulmonary concentrations of the active moiety as a function of the route of administration in critically ill patients have not been precisely documented. In this study, CMS and colistin concentrations were measured on two separate occasions within the plasma and epithelial lining fluid (ELF) of critically ill patients (n = 12) who had received 2 million international units (MIU) of CMS by aerosol delivery and then intravenous administration. The pharmacokinetic analysis was conducted using a population approach and completed by pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulations. The ELF colistin concentrations varied considerably (9.53 to 1,137 mg/liter), but they were much higher than those in plasma (0.15 to 0.73 mg/liter) after aerosol delivery but not after intravenous administration of CMS. Following CMS aerosol delivery, typically, 9% of the CMS dose reached the ELF, and only 1.4% was presystemically converted into colistin. PK-PD analysis concluded that there was much higher antimicrobial efficacy after CMS aerosol delivery than after intravenous administration. These new data seem to support the use of aerosol delivery of CMS for the treatment of pulmonary infections in critical care patients. PMID:25267660

  4. A proposal for a novel rationale for critical effect size in dose-response analysis based on a multi-endpoint in vivo study with methyl methanesulfonate.

    PubMed

    Zeller, Andreas; Tang, Leilei; Dertinger, Stephen D; Funk, Juergen; Duran-Pacheco, Gonzalo; Guérard, Melanie

    2016-05-01

    Methyl methanesulfonate, a well-known direct-acting genotoxicant, was assessed in a multi-endpoint study in rats using six closely spaced dose levels. The main goal of the study was to investigate the genotoxic response at very low doses and to analyse this response with dedicated statistical tools in order to find a Point of Departure (PoD) and related metrics. Software packages like PROAST or EPA-BMDS require the toxicologist to define a so-called critical effect size (CES) or benchmark response (BMR) and this choice has a large impact on the result of the PoD calculation. Currently, increases of 5%, 10% or 1 standard deviation over concurrent vehicle controls have been proposed for CES/BMR, values that may or may not be suited for all genotoxicity endpoints. Based on the data obtained in this study, we propose an endpoint specific CES approach that reflects the typical evaluation process of a regulatory acceptable genotoxicology study. However, we are aware that this ratio-based CES strategy will need to be more fully developed with additional experimentation and should be mainly seen as a starting point for scientific discussion. PMID:26590612

  5. 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

  6. 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

  7. Characterization of the second prosthetic group of the flavoenzyme NADH-acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Colby, J; Dalton, H

    1979-01-01

    1. A new two-step purification is described that routinely yields 100mg quantities of component C for biochemical studies. 2. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of iron, 2 mol of acid-labile sulphide (S) and 1 mol of FAD per mol of protein. 3. The Fe-S core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50mM-Tris/HCl buffer, pH 8.5 (4:1, v/v), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S(S-Cys)4] centre per mol of protein. 4. E.p.r. spectroscopy confirms the presence of a Fe-S centre in component C. 5. Component C catalyses the reduction by NADH of ferricyanide, 2,6-dichlorophenol-indophenol or horse heart cytochrome c, with specific activities of 50--230 units/mg of protein. 6. The optimum pH for the NADH-acceptor reductase activity is 8.5--9.0, and the apparent Km values for NADH and NADPH are 0.05mM and 15.5mM respectively. 7. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene. PMID:220953

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate

    SciTech Connect

    Lindler, L.E.; Macrina, F.L.

    1987-07-01

    We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci.

  15. The Arabidopsis transcription factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 is a direct substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and regulates immunity.

    PubMed

    Kang, Sining; Yang, Fan; Li, Lin; Chen, Huamin; Chen, She; Zhang, Jie

    2015-03-01

    Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1(SSAA)) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1(SSAA) does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation. PMID:25609555

  16. Effect of methyl methanesulfonate on hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9

    PubMed Central

    Kumar, Vineet; Ara, Gulshan; Afzal, Mohammad; Siddique, Yasir Hasan

    2011-01-01

    Methyl methanesulfonate (MMS) is an anti-carcinogenic drug and its toxicity has been reported in various experimental models. The hsp70s are a family of ubiquitously expressed heat shock proteins. In the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals. Nowadays emphasis is given to the use of alternatives to mammals in testing, research and education. The European Centre for the Validation of Alternative Methods (EVCAM) has recommended the use of Drosophila as an alternative model for scientific studies. Almost all living organisms possess proteins with a similar structure to that of hsp70s. In the present study, the toxicity of MMS was evaluated by quantifying hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9, at different doses and hours of exposure. We studied the effect of 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS at 2, 4, 24 and 48 hours of exposure on hsp70 expression by using the soluble O-nitrophenyl-β-D-galactopyranoside (ONPG) assay and on establishing the tissue damage by the Trypan blue exclusion assay in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9. A dose-dependent increase in the expression of hsp70 was observed at 0.25, 0.50, and 0.75 µl/ml of MMS compared to the control. At the highest dose, i.e. 1.0 µl/ml of MMS, the activity of hsp70 was decreased due to tissue damage. PMID:22058658

  17. Suppression of aflatoxin B1- or methyl methanesulfonate-induced chromosome aberrations in rat bone marrow cells after treatment with S-methyl methanethiosulfonate.

    PubMed

    Ito, Y; Nakamura, Y; Nakamura, Y

    1997-10-24

    The suppressive effect of S-methyl methanethiosulfonate (MMTS) on aflatoxin B1 (AFB1)- or methyl methanesulfonate (MMS)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. MMTS significantly suppressed CA induced by both AFB1 (an indirect-acting carcinogen) and MMS (a direct-acting carcinogen). Suppression was observed at all periods (6, 12, 18, 24 and 48 h) after AFB1 or MMS treatment and in all doses of AFB1 (5, 10 and 20 mg/kg) or MMS (50, 75 and 100 mg/kg) investigated. AFB1-induced CA was potently suppressed by MMTS given between 2 h before and 6 h after the AFB1 injection. The suppression of AFB1-induced CA by MMTS paralleled the dose of MMTS when MMTS was given in a dose range of 1-20 mg/kg body weight. MMS-induced CA was potently suppressed by MMTS given between 2 h before and 2 h after the MMS injection. The suppressive effect of MMTS on MMS-induced CA paralleled the dose of MMTS when MMTS was given in a dose range of 1-15 mg/kg body weight. Diphenyl disulfide, which modifies -SH groups in proteins like MMTS, also significantly suppressed both AFB1- and MMS-induced CA. Although other mechanisms are not excluded, the suppression of carcinogen-induced CA by MMTS may result from the ability of MMTS to modify -SH groups in proteins. The juices of cabbage and onion, which contain considerable amounts of MMTS and S-methyl-L-cysteinesulfoxide (the precursor of MMTS), also significantly suppressed AFB1- or MMS-induced CA. These results suggest that MMTS is a possible chemopreventive agent against cancer. PMID:9393623

  18. Investigating the 'Iron Hypothesis' in the North Pacific: Trans-Pacific Dust and Methanesulfonate (MSA) in the Denali Ice Core, Alaska

    NASA Astrophysics Data System (ADS)

    Saylor, P. L.; Osterberg, E. C.; Winski, D.; Ferris, D. G.; Koffman, B. G.; Kreutz, K. J.; Wake, C. P.; Campbell, S. W.

    2015-12-01

    Oceanic deposition of Asian-sourced, Iron-rich dust particulate has been linked to enhanced phytoplankton productivity in regions of the Pacific Ocean. High Nutrient Low Chlorophyll (HNLC) ocean regions, such as the North Pacific, are hypothesized to play a significant role in changing atmospheric CO­2 concentrations on glacial-interglacial timescales. Phytoplankton blooms generate methanesulfonate (MSA), an atmospheric oxidation product of dimethylsulfide (DMS) that is readily aerosolized and deposited in nearby glacial ice. In the summer of 2013, an NSF-funded team from Dartmouth College and the Universities of Maine and New Hampshire collected two 1000 year-long parallel ice cores to bedrock from the summit plateau of Mount Hunter in Denali National Park, Alaska (62.940° N, 151.088° W, 3912 m elevation). The Mt. Hunter ice core site is well situated to record changes in trans-Pacific dust flux and MSA emissions in the North Pacific. Here we investigate the history of dust flux to Denali over the last millennium using major and trace element chemistry and microparticle concentration and size distribution data from the Mt. Hunter cores. We evaluate potential controlling mechanisms on Denali dust flux including conditions at Asian dust sources (storminess, wind speed, precipitation), the strength of the Aleutian Low, and large-scale climate modes such as the El Niño-Southern Oscillation and the Pacific Decadal Oscillation. We also evaluate the Mt. Hunter record for relationships between dust flux and MSA concentrations to investigate whether dust fertilization enhanced North Pacific phytoplankton production over the past 1000 years. Future work will create a composite North Pacific dust record using new and existing Mt. Logan ice core records to evaluate these relationships over the entire Holocene.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  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. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. Expression analysis of two P450 monooxygenase genes of the tobacco cutworm moth (Spodoptera litura) at different developmental stages and in response to plant allelochemicals.

    PubMed

    Wang, Rui-Long; Li, Jun; Staehelin, Christian; Xin, Xiao-Wei; Su, Yi-Juan; Zeng, Ren-Sen

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) of insects are known to be involved in the metabolism or detoxification of plant allelochemicals and insecticides. Spodoptera litura (Lepidoptera, Noctuidae) is a polyphagous moth responsible for severe yield losses in many crops. In this study, two full-length P450 genes, CYP6B48 and CYP6B58, were cloned from S. litura. The cDNA sequences encode proteins with 503 and 504 amino acids, respectively. Phylogenetic analysis revealed that CYP6B48 and CYP6B58 belong to the CYP6B subfamily of P450s. Quantitative real-time PCR analyses showed that CYP6B48 and CYP6B58 were expressed only at larval stage, but not at pupal and adult stages. The highest levels of transcripts were found in the midguts and fat bodies of the larvae. No expression was detected in the ovary or hemolymph. Feeding with diets containing cinnamic acid, quercetin, or coumarin did not affect expression of CYP6B48. In contrast, diet supplemented with xanthotoxin dramatically increased the levels of CYP6B48 transcript in the midgut and fat bodies. Larvae fed with flavone had high levels of transcript of CYP6B48 in the midgut, whereas only slightly elevated levels were found in the fat bodies. Effects of the tested allelochemicals on CYP6B58 expression were minor. Hence, our findings show that S. litura responds to specific allelochemicals such as xanthotoxin with the accumulation of CYP6B48 transcripts, suggesting that specific signals in the food control the insect's ability to convert toxic allelochemicals to less harmful forms at the transcriptional level. PMID:25547988

  5. avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus.

    PubMed Central

    Yu, J; Chang, P K; Cary, J W; Bhatnagar, D; Cleveland, T E

    1997-01-01

    Recent studies have shown that at least 17 genes involved in the aflatoxin biosynthetic pathway are clustered within a 75-kb DNA fragment in the genome of Aspergillus parasiticus. Several additional transcripts have also been mapped to this gene cluster. A gene, avnA (previously named ord-1), corresponding to one of the two transcripts identified earlier between the ver-1 and omtA genes on the gene cluster was sequenced. The nucleotide sequence of the avnA gene contains a coding region for a protein of 495 amino acids with a calculated molecular mass of 56.3 kDa. The gene consists of three exons and two introns. Disruption of the avnA gene in the wild-type aflatoxigenic A. parasiticus strain (SU1-N3) resulted in a nonaflatoxigenic mutant which accumulated a bright yellow pigment. Thin-layer chromatographic studies with six different solvent systems showed that the migration patterns of the accumulated metabolite were identical to those of averantin, a known aflatoxin precursor. Precursor feeding studies with this mutant showed that norsolorinic acid and averantin were not converted to aflatoxin whereas 5'-hydroxyaverantin, averufanin, averufin, versicolorin A. sterigmatocystin, and O-methylsterigmatocystin were converted to aflatoxins. Southern blot analysis of the wild-type strain and avnA-disrupted mutant strain indicated that the avnA gene was disrupted in the mutant strain. A search of the GenBank database for similarity indicated that the avnA gene encodes a cytochrome P-450-type monooxygenase, and it has been assigned to a new P-450 gene family named CYP60A1. We have therefore concluded that the avnA gene encodes a fungal cytochrome P-450-type enzyme which is involved in the conversion of averantin to averufin in the aflatoxin biosynthetic pathway in A. parasiticus. PMID:9097431

  6. Effects of lytic polysaccharide monooxygenase oxidation on cellulose structure and binding of oxidized cellulose oligomers to cellulases.

    PubMed

    Vermaas, Josh V; Crowley, Michael F; Beckham, Gregg T; Payne, Christina M

    2015-05-21

    In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of β-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending on enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce steric effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases with

  7. 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

  8. Electrodeposited nanostructured lead dioxide as a thin film electrode for a lightweight lead-acid battery

    NASA Astrophysics Data System (ADS)

    Egan, D. R. P.; Low, C. T. J.; Walsh, F. C.

    Thin films of nanostructured lead dioxide are investigated as a positive electrode material for a lightweight lead-acid battery. The films are obtained by constant current deposition from electrolytes of lead methanesulfonate in methanesulfonic acid. The films are tested in two conditions namely (a) cyclic voltammetry and (b) constant current battery cycling in sulfuric acid. The charge and discharge current density, charge density and charge efficiency are measured as a function of cycle number. The effect of deposition conditions, such as solution temperature (295 and 333 K), type of substrate and electrolyte additive (hexadecyltrimethylammonium hydroxide), on the electrochemical performance of the PbO 2 in sulfuric acid is investigated. It is found that the as-deposited lead dioxide film is compact and nanostructured β-phase structure. Following successive cycling in sulfuric acid, the compact thin film gradually transforms into a porous microstructure consisting of positive active material (PbO 2 and PbSO 4), several tens of nanometres size. The charge density, discharge density and peak discharge current density of the PbO 2 improve with cycling of the thin film electrode.

  9. SmoXYB1C1Z of Mycobacterium sp. strain NBB4: a soluble methane monooxygenase (sMMO)-like enzyme, active on C2 to C4 alkanes and alkenes.

    PubMed

    Martin, Kiri E; Ozsvar, Jazmin; Coleman, Nicholas V

    2014-09-01

    Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc(2)-155. Cells of mc(2)-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc(2)-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc(2)-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc(2)-155(pSmo) provides a new model for studying sMMO-like monooxygenases. PMID:25015887

  10. SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C2 to C4 Alkanes and Alkenes

    PubMed Central

    Martin, Kiri E.; Ozsvar, Jazmin

    2014-01-01

    Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc2-155. Cells of mc2-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc2-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc2-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc2-155(pSmo) provides a new model for studying sMMO-like monooxygenases. PMID:25015887

  11. Characterization of the two-component, FAD-dependent monooxygenase SgcC that requires carrier protein-tethered substrates for the biosynthesis of the enediyne antitumor antibiotic C-1027.

    PubMed

    Lin, Shuangjun; Van Lanen, Steven G; Shen, Ben

    2008-05-21

    C-1027 is a potent antitumor antibiotic composed of an apoprotein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the biosynthesis of which from l-alpha-tyrosine requires five proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth protein, SgcC5, catalyzes the incorporation of this beta-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (peptidyl carrier protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O 2 and FADH 2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted beta-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but does not accept 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. Together with the in vitro data for SgcC4, SgcC1, and SgcC3, the results establish that SgcC catalyzes the hydroxylation of ( S)-3-chloro-beta-tyrosyl-S-SgcC2 as the final step in the biosynthesis of the ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety prior to incorporation into C-1027. SgcC now represents the first biochemically characterized two-component, FAD-dependent monooxygenase that acts on a carrier-protein-tethered aromatic substrate. PMID:18426211

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

    PubMed Central

    Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J.; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

    2015-01-01

    Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

  20. Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria ▿

    PubMed Central

    Furuya, Toshiki; Hirose, Satomi; Semba, Hisashi; Kino, Kuniki

    2011-01-01

    The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ54 factor, a gene encoding the σ54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. PMID:21856847

  1. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes.

    PubMed

    Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

    2015-01-01

    Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD-coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation-were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria-Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

  2. Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M.

    PubMed

    Vasil'ev, V I; Tikhonova, T V; Gvozdev, R I; Tukhvatullin, I A; Popov, V O

    2006-12-01

    The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out. PMID:17223785

  3. The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis

    PubMed Central

    Sciara, Giuliano; Kendrew, Steven G.; Miele, Adriana E.; Marsh, Neil G.; Federici, Luca; Malatesta, Francesco; Schimperna, Giuliana; Savino, Carmelinda; Vallone, Beatrice

    2003-01-01

    ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 Å) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O2/H2O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme–substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure. PMID:12514126

  4. Crystal Structure of Baeyer−Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway

    SciTech Connect

    Beam, Miranda P.; Bosserman, Mary A.; Noinaj, Nicholas; Wehenkel, Marie; Rohr, Jurgen; Kentucky

    2009-06-01

    Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV's structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9 A. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments identifies several residues that participate in cofactor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis.

  5. Monooxygenase activity and contaminant burdens of pipping heron embryos in Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.

    1991-01-01

    Black-crowned night-heron (Nvcticorax nvcticorax) pipping embryos were studied from undisturbed (Chincoteague National Wildl ife Refuge, VA) and industrialized (Cat Island, Green Bay WI, and Bair and W. Marin Islands, San Francisco Bay, CA) locations. Hepatic aryl hydrocarbon hydroxylase (AHH) , ethoxyresorufin-O-dealkylase, (EROD), benzyloxyROD (BROD), pentoxyROD (PROD) and ethoxycoumarinOD (ECOD) activities and burdens of organochlorines (embryo + yolk sac - liver) were quantified. AHH, BROD, ECOD and EROD were induced up to 100-fold (P<.O5) in embryos from Cat Island compared to the other sites. Greatest burdens of total PCBs and p,p?DDE were detected in Cat Island embryos. Monooxygenase activities (AHH, BROD, ECOD and EROD) and PCB concentrations were significantly correlated (r=O.50 to 0.72). These and other data indicate that monooxygenases may be rapid and inexpensive biomarkers of exposure to some PCB congeners. Current efforts include determination of PCB congeners and other contaminants in these embryos, additional characterization of the induced P-450 isozymes, and expanding the study to include heron embryos and nestlings at other estuaries.

  6. Directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone.

    PubMed

    Parra, Loreto P; Acevedo, Juan P; Reetz, Manfred T

    2015-07-01

    Phenylacetone monooxygenase (PAMO) is an exceptionally robust Baeyer-Villiger monooxygenase, which makes it ideal for potential industrial applications. However, its substrate scope is limited, unreactive cyclohexanone being a prominent example. Such a limitation is unfortunate, because this particular transformation in an ecologically viable manner would be highly desirable, the lactone and the respective lactam being of considerable interest as monomers in polymer science. We have applied directed evolution in search of an active mutant for this valuable C-C activating reaction. Using iterative saturation mutagenesis (ISM), several active mutants were evolved, with only a minimal trade-off in terms of stability. The best mutants allow for quantitative conversion of 2 mM cyclohexanone within 1 h reaction time. In order to circumvent the NADP(+) regeneration problem, whole E. coli resting cells were successfully applied. Molecular dynamics simulations and induced fit docking throw light on the origin of enhanced PAMO activity. The PAMO mutants constitute ideal starting points for future directed evolution optimization necessary for an industrial process. PMID:25675885

  7. Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium.

    PubMed

    Hu, Jie; Okawa, Hiroto; Yamamoto, Kentaro; Oyama, Kazuhiko; Mitomi, Masaaki; Anzai, Hiroyuki

    2011-03-01

    Pyripyropenes are potent inhibitors of acyl-CoA:cholesterol acyltransferase, which were initially discovered to be produced by Aspergillus fumigatus. Recently, Penicillium coprobium PF1169 has also found to produce pyripyropene A (PyA), which exhibits insecticidal properties. Pyripyropenes are natural hybrid products of both terpenoid and polyketide origin. In our research, based on data generated using the Genome Sequencer FLX for P. coprobium PF1169, we predicted the biosynthetic gene cluster of PyA by blast analysis comparing with polyketide synthase and prenyltransferase of other species. By screening the genomic fosmid library, nine open reading frames (ppb1 to ppb9) related to the biosynthesis of PyA were deduced. Among them, two cytochrome P450 monooxygenase genes (ppb3 and ppb4) were separately introduced into the model fungus A. oryzae. Bioconversion of certain predicted intermediates in the transformants has elucidated the manner of hydroxylation in the biosynthetic pathway by the expressed products of these two genes (P450-1 and P450-2). That is, P450-1 exhibits monooxygenase activity and plays the hydroxylation role at C-11 of pyripyropene E. While P450-2 plays an active role in the hydroxylation of C-7 and C-13 of pyripyropene O. PMID:21224862

  8. Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath.

    PubMed

    Ali, Hanif; Murrell, J Colin

    2009-03-01

    A series of integrative and versatile broad-host-range promoter-probe vectors carrying reporter genes encoding green fluorescent protein (GFP), catechol 2,3-dioxygenase (XylE) or beta-galactosidase (LacZ) were constructed for use in methanotrophs. These vectors facilitated the measurement of in vivo promoter activity in methanotrophs under defined growth conditions. They were tested by constructing transcriptional fusions between the soluble methane monooxygenase (sMMO) sigma(54) promoter or particulate methane monooxygenase (pMMO) sigma(70) promoter from Methylococcus capsulatus and the reporter genes. Reporter gene activity was measured under high- and low-copper growth conditions and the data obtained closely reflected transcriptional regulation of the sMMO or pMMO operon, thus demonstrating the suitability of these vectors for assessing promoter activity in methanotrophs. When beta-galactosidase expression was coupled with the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide it yielded a sensitive and powerful screening system for detecting cells expressing this reporter gene. These data were substantiated with independent experiments using RT-PCR and RNA dot-blot analysis. PMID:19246747

  9. Crystal Structure of Baeyer-Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway†

    PubMed Central

    Beam, Miranda P.; Bosserman, Mary A.; Noinaj, Nicholas; Wehenkel, Marie; Rohr, Jürgen

    2009-01-01

    Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kD homo-dimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV’s structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9Å. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments implicate several residues to participate in co-factor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis. PMID:19364090

  10. Crystal structure of Baeyer-Villiger monooxygenase MtmOIV, the key enzyme of the mithramycin biosynthetic pathway .

    PubMed

    Beam, Miranda P; Bosserman, Mary A; Noinaj, Nicholas; Wehenkel, Marie; Rohr, Jürgen

    2009-06-01

    Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV's structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9 A. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments identifies several residues that participate in cofactor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis. PMID:19364090

  11. Molecular cloning and xenobiotic induction of seven novel cytochrome P450 monooxygenases in Aedes albopictus.

    PubMed

    Chan, Hiang Hao; Wajidi, Mustafa Fadzil Farid; Zairi, Jaal

    2014-01-01

    Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450. PMID:25399430

  12. 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

  13. Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

    PubMed Central

    Lontoh, Sonny; Semrau, Jeremy D.

    1998-01-01

    Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (Vmax) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (Ks) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, Vmax/Ks, doubled from 4.9 × 10−3 to 9.9 × 10−3 liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the Vmax for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the Vmax to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in Ks from 36 to 7.9 μM. Vmax/Ks for TCE degradation by pMMO also increased from 6.9 × 10−5 to 5.2 × 10−4 liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO. PMID:16349516

  14. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  15. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration. PMID:19560175

  16. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection is Associated with Induction of Flavin-containing Monooxygenase-3 (FMO3) in Hepatocytes

    EPA Science Inventory

    Acetaminophen (APAP) pretreatment with a low hepatotoxic dose in mice results in resistance to a second, higher dose of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in...

  17. Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

    PubMed Central

    Xu, Y; Mortimer, M W; Fisher, T S; Kahn, M L; Brockman, F J; Xun, L

    1997-01-01

    Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation. PMID:9023192

  18. 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...

  19. EXPRESSION OF BRANCHIAL FLAVIN-CONTAINING MONOOXYGENASE IS DIRECTLY CORRELATED WITH SALINITY-INDUCED ALDICARB TOXICITY IN THE EURYHALINE FISH (ORYZIAS LATIPES). (R826109)

    EPA Science Inventory

    Abstract

    Earlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioact...

  20. Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

    PubMed Central

    Jawallapersand, Poojah; Mashele, Samson Sitheni; Kovačič, Lidija; Stojan, Jure; Komel, Radovan; Pakala, Suresh Babu; Kraševec, Nada; Syed, Khajamohiddin

    2014-01-01

    Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative

  1. Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification.

    PubMed

    Lloyd, J S; Bhambra, A; Murrell, J C; Dalton, H

    1997-08-15

    The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated forms, B' and B". Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B. This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15,852.6+/-0.4 Da and 14,629.5+/-0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N-terminus of protein B. This truncate was designated B" and had a molecular mass of 12,709.93+/-0.02 Da. Proteins B' and B" were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation. Expression of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from E. coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild-type protein B. However, ESI-MS confirmed that protein B' was still present. Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations. ESI-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell. PMID:9310362

  2. Metabolism of fluperlapine by cytochrome P450-dependent and flavin-dependent monooxygenases in continuous cultures of rat and human cells.

    PubMed

    Fischer, V; Wiebel, F J

    1990-04-15

    The metabolism of fluperlapine, a neuroleptic dibenzazepine derivative with a N-methyl-piperazinyl substituent, was investigated in continuous cultures of rat and human cells which express various cytochrome P450-dependent monooxygenase activities. The differentiated rat hepatoma cells H4IIEC3/G- and their variants 2sFou and FGC-5 metabolized fluperlapine predominantly by N-oxygenation and only to a minor degree by N-demethylation or glucuronidation of primary phenolic products. Total fluperlapine metabolism in dedifferentiated rat hepatoma cells H5 and partially differentiated human hepatoma cells HepG2 was much smaller than in the differentiated rat hepatoma lines. This was primarily attributable to their low capacity for N-oxygenation. Human lung adenocarcinoma lines NCI-H322 and NCI-H358 formed only trace amounts of fluperlapine N-oxide. Pretreatment of 2sFou cells with benz(a)anthracene, phenobarbital or dexamethasone markedly increased the formation of N-demethylated and glucuronidated products but did not affect the rate of N-oxide formation. Guanethidine and cysteamine, inhibitors of flavin-dependent monooxygenase activity, reduced fluperlapine N-oxidation more strongly than aldrin epoxidation, a marker for cytochrome P450 activity. In contrast, n-octylamine inhibited aldrin epoxidation but was without effect on fluperlapine N-oxygenation. The results suggest that certain cells in continuous culture are capable of expressing flavin-dependent monooxygenase(s) in addition to cytochrome P450-containing monooxygenases. Such cells may offer useful systems for studying the oxidative metabolism of a broad spectrum of xenobiotics and analysing the importance of the two oxygenation reactions for the biological effects of their substrates. PMID:2322314

  3. Production of ω-hydroxy octanoic acid with Escherichia coli.

    PubMed

    Kirtz, Marko; Klebensberger, Janosch; Otte, Konrad B; Richter, Sven M; Hauer, Bernhard

    2016-07-20

    The present proof-of-concept study reports the construction of a whole-cell biocatalyst for the de novo production of ω-hydroxy octanoic acid. This was achieved by hijacking the natural fatty acid cycle and subsequent hydroxylation using a specific monooxygenase without the need for the additional feed of alkene-like precursors. For this, we used the model organism Escherichia coli and increased primarily the release of the octanoic acid precursors by overexpressing the plant thioesterase FatB2 from Cuphea hookeriana in a β-oxidation deficient strain, which lead to the production of 2.32mM (8.38mggcww(-1)) octanoic acid in 24h. In order to produce the corresponding ω-hydroxy derivative, we additionally expressed the engineered self-sufficient monooxygenase fusion protein CYP153AMaq(G307A)-CPRBM3 within the octanoic acid producing strain. With this, we finally produced 234μM (0.95mggcww(-1)) ω-hydroxy octanoic acid in a 20h fed-batch set-up. PMID:27184430

  4. Comparison of somatic reversions between the ivory allele and transposon-caused mutant alleles at the white locus of Drosophila melanogaster after larval treatment with X rays and ethyl methanesulfonate

    SciTech Connect

    Ryo, H.; Yoo, M.A.; Fujikawa, K.; Kondo, S.

    1985-07-01

    Somatic reversion of strains with the ivory (wi) allele, a mutation associated with a tandem duplication of a DNA sequence at the white locus, increased with the age of larvae at the time of X-irradiation as expected from the increase in the number of target cells. In contrast, two independently isolated strains with unstable w+ loci associated with insertion of transposable elements showed higher reversion frequencies after treatment with X rays or ethyl methanesulfonate (EMS) at early larval stages than at late stages. Nevertheless, both the wi strain and the two unstable w+ strains reverted at nearly equal rates after treatment with X rays or EMS at early larval stages. Possible similarity in hot spot structure for the high reversibility of the two types of mutations is discussed in relation to production of presumed mutator-type cofactors specific to the transposon-caused mutations at early larval stages.

  5. Mechanistic studies of the reaction of reduced methane monooxygenase hydroxylase with dioxygen and substrates

    SciTech Connect

    Valentine, A.M.; Stahl, S.S.; Lippard, S.J.

    1999-04-28

    Soluble methane monooxygenase (sMMO) catalyzes the oxidation of methane to methanol. Single-turnover reactions of sMMO from Methylococcus capsulatus (Bath) were studied by stopped-flow optical spectroscopy to examine further the activated dioxygen intermediates and their reactions with hydrocarbon substrates. A diiron(III) peroxo species designated H{sub peroxo} is the first intermediate observed in the reaction between the chemically reduced hydroxylase (H{sub red}) and dioxygen. The optical spectrum of this species determined by diode array detection is presented for the first time and exhibits visible absorption bands with {lambda}{sub max} {approx} 420 nm ({epsilon} = 4,000 M{sup {minus}1} cm{sup {minus}1}) and {lambda}{sub max} = 725 nm ({epsilon} = 1,800 M{sup {minus}1} cm{sup {minus}1}). The temperature dependences of the rate constants for formation and decay of H{sub peroxo} and for the subsequent intermediate, Q, were examined in the absence and in the presence of hydrocarbon substrates, and activation parameters for these reactions were determined. For single-turnover reaction kinetics monitored at 420 nm, the {lambda}{sub max} for Q, a nonlinear Eyring plot was obtained when acetylene or methane was present in sufficiently high concentration. This behavior reflects a two-step mechanism, Q formation followed by Q decay, in which the rate-determining step changes depending on the temperature. The rate of H{sub peroxo} formation does not depend on dioxygen concentration, indicating that an effectively irreversible step involving dioxygen precedes formation of the diiron(III) peroxo species. The rate constant observed at 4 C for H{sub peroxo} formation, 1--2 s{sup {minus}1}, is slower than that determined previously by Moessbauer and optical spectroscopy, {approximately}20--25 s{sup {minus}1} (Liu, K. E., et al. J. Am. Chem. Soc. 1995, 117, 4997--4998; 10174--10185). Possible explanations for this discrepancy include the existence of two distinct peroxo

  6. Localization of human flavin-containing monooxygenase genes FMO2 and FMO5 to chromosome 1q

    SciTech Connect

    McCombie, R.R.; Shephard, E.A.; Dolphin, C.T.

    1996-06-15

    The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1 to FMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3, and FMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human-rodent somatic cell hybrids demonstrates that the two remaining identified members of the FMO gene family, FMO2 and FMO5, also are located on chromosome 1q. 19 refs., 1 fig., 1 tab.

  7. Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family.

    PubMed

    Morgenstern, Ingo; Powlowski, Justin; Tsang, Adrian

    2014-11-01

    Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. PMID:25217478

  8. Enantioselective Benzylic Hydroxylation Catalysed by P450 Monooxygenases: Characterisation of a P450cam Mutant Library and Molecular Modelling.

    PubMed

    Eichler, Anja; Gricman, Łukasz; Herter, Susanne; Kelly, Paul P; Turner, Nicholas J; Pleiss, Jürgen; Flitsch, Sabine L

    2016-03-01

    Cytochrome P450 monooxygenases can catalyse the stereoselective C-H activation of a very broad range of substrates. Prediction and control of enantioselectivity of this enzyme class is of great interest for the synthesis of high-value chiral molecules. Here we have used a combination of molecular dynamics simulations and experimental screening to study the enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene. Small variations either in substrate structure or in enzyme active site architecture were shown to lead to dramatic changes in enantioselectivity; this was broadly in agreement with computational predictions. In addition to validating computational approaches, these studies have provided us with a deeper understanding of effects that might control stereoselectivity in these biooxidation reactions. PMID:26698167

  9. Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family

    PubMed Central

    Powlowski, Justin; Tsang, Adrian

    2014-01-01

    Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. PMID:25217478

  10. 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

  11. 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.

  12. Single Turnover Reveals Oxygenated Intermediates in Toluene/o-Xylene Monooxygenase in the Presence of the Native Redox Partners

    PubMed Central

    Liang, Alexandria Deliz; Lippard, Stephen J.

    2015-01-01

    Toluene/o-xylene monooxygenase (ToMO) is a non-heme diiron protein that activates O2 for subsequent arene oxidation. ToMO utilizes four protein components, a catalytic hydroxylase (ToMOH), a regulatory protein (To-MOD), a Rieske protein (ToMOC), and a reductase (ToMOF). Here, we examine O2 activation and substrate hydroxylation in the presence of all four-protein components. Studies of this system demonstrate the importance of ToMOC for formation of the active hydroxylating species that are not revealed when dithionite and mediators are used as the reductant. This reactivity is discussed in light of other O2-activating, non-heme diiron enzymes. PMID:26267757

  13. Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015

    PubMed Central

    Zhang, Chunxiao; Sheng, Chaolan; Wang, Wei; Hu, Hongbo; Peng, Huasong; Zhang, Xuehong

    2015-01-01

    Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces. PMID:26305803

  14. Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

    PubMed

    Bahadoor, Adilah; Schneiderman, Danielle; Gemmill, Larissa; Bosnich, Whynn; Blackwell, Barbara; Melanson, Jeremy E; McRae, Garnet; Harris, Linda J

    2016-01-22

    A second structural gene required for culmorin biosynthesis in the plant pathogen Fusarium graminearum is described. Clm2 encodes a regio- and stereoselective cytochrome P450 monooxygenase for C-11 of longiborneol (1). Clm2 gene disruptants were grown in liquid culture and assessed for culmorin production via HPLC-evaporative light scattering detection. The analysis indicated a complete loss of culmorin (2) from the liquid culture of the ΔClm2 mutants. Culmorin production resumed in a ΔClm2 complementation experiment. A detailed analysis of the secondary metabolites extracted from the large-scale liquid culture of disruptant ΔClm2D20 revealed five new natural products: 3-hydroxylongiborneol (3), 5-hydroxylongiborneol (4), 12-hydroxylongiborneol (5), 15-hydroxylongiborneol (6), and 11-epi-acetylculmorin (7). The structures of the new compounds were elucidated by a combination of HRMS, 1D and 2D NMR, and X-ray crystallography. PMID:26673640

  15. Copper-Dioxygen Complex Mediated C-H Bond Oxygenation: Relevance for Particulate Methane Monooxygenase (pMMO)

    PubMed Central

    Himes, Richard A.; Karlin, Kenneth D.

    2009-01-01

    Summary Particulate methane monooxygenase (pMMO), an integral membrane protein found in methanotrophic bacteria, catalyzes the oxidation of methane to methanol. Expression and greater activity of the enzyme in the presence of copper ion suggest that pMMO is a cuprous metalloenzyme. Recent advances – especially the first crystal structures of pMMO – have energized the field, but the nature of the active site(s) and the mechanism of methane oxidation remain poorly understood – yet hotly contested. Herein the authors briefly review the current understanding of the pMMO metal sites, and discuss advances in small molecule Cu-O2 chemistry that may contribute to an understanding of copper-ion mediated hydrocarbon oxidation chemistry. PMID:19286415

  16. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases

    PubMed Central

    Kjaergaard, Christian H.; Qayyum, Munzarin F.; Wong, Shaun D.; Xu, Feng; Hemsworth, Glyn R.; Walton, Daniel J.; Young, Nigel A.; Davies, Gideon J.; Walton, Paul H.; Johansen, Katja Salomon; Hodgson, Keith O.; Hedman, Britt; Solomon, Edward I.

    2014-01-01

    Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9–11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy. PMID:24889637

  17. Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer-Villiger and sulfide oxidations.

    PubMed

    Zambianchi, F; Pasta, P; Carrea, G; Colonna, S; Gaggero, N; Woodley, J M

    2002-06-01

    The performance, in Baeyer-Villiger and heteroatom oxidations, of a partially purified preparation of cyclohexanone monooxygenase obtained from an Escherichia coli strain in which the gene of the enzyme was cloned and overexpressed was investigated. As model reactions, the oxidations of racemic bicyclo[3.2.0]hept-2-en-6-one into two regioisomeric lactones and of methyl phenyl sulphide into the corresponding (R)-sulphoxide were used. Enzyme stability and reuse, substrate and product inhibition, product removal, and cofactor recycling were evaluated. Of the various NADPH regeneration systems tested, 2-propanol/alcohol dehydrogenase from Thermoanerobium brockii appeared the most suitable because of the low cost of the second substrate and the high regeneration rate. Concerning enzyme stability, kosmotropic salts were the only additives able to improve it (e.g., half-life from 1 day in diluted buffer to 1 week in 1 M sodium sulphate) but only under storage conditions. Instead, significant stabilization under working conditions was obtained by immobilization on Eupergit C (half-life approximately 2.5 days), a procedure that made it possible to reuse the catalyst up to 16 times with complete substrate (5 g x L(-1)) conversion at each cycle. Reuse of free enzyme was also achieved in a membrane reactor but with lower efficiency. Water-organic solvent biphasic systems, which would overcome substrate inhibition and remove from the aqueous phase, where reaction takes place, the formed product, were unsuccessful because of their destabilizing effect on cyclohexanone monooxygenase. More satisfactory was continuous substrate feeding, which shortened reaction times and, very importantly, yielded in the case of bicyclo[3.2.0]hept-2-en-6-one (10 g x L(-1)) both lactone products with high optical purity (enantiomeric excess > or = 96%), which was not the case when all of the substrate was added in a single batch. PMID:12115117

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

    PubMed Central

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

    2014-01-01

    Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells. PMID:23994590

  19. Structural biology of redox partner interactions in P450cam monooxygenase: a fresh look at an old system.

    PubMed

    Sevrioukova, Irina F; Poulos, Thomas L

    2011-03-01

    The P450cam monooxygenase system consists of three separate proteins: the FAD-containing, NADH-dependent oxidoreductase (putidaredoxin reductase or Pdr), cytochrome P450cam and the 2Fe2S ferredoxin (putidaredoxin or Pdx), which transfers electrons from Pdr to P450cam. Over the past few years our lab has focused on the interaction between these redox components. It has been known for some time that Pdx can serve as an effector in addition to its electron shuttle role. The binding of Pdx to P450cam is thought to induce structural changes in the P450cam active site that couple electron transfer to substrate hydroxylation. The nature of these structural changes has remained unclear until a particular mutant of P450cam (Leu358Pro) was found to exhibit spectral perturbations similar to those observed in wild type P450cam bound to Pdx. The crystal structure of the L358P variant has provided some important insights on what might be happening when Pdx docks. In addition to these studies, many Pdx mutants have been analyzed to identify regions important for electron transfer. Somewhat surprisingly, we found that Pdx residues predicted to be at the P450cam-Pdx interface play different roles in the reduction of ferric P450cam and the ferrous P450-O(2) complex. More recently we have succeeded in obtaining the structure of a chemically cross-linked Pdr-Pdx complex. This fusion protein represents a valid model for the noncovalent Pdr-Pdx complex as it retains the redox activities of native Pdr and Pdx and supports monooxygenase reactions catalyzed by P450cam. The insights gained from these studies will be summarized in this review. PMID:20816746

  20. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

    PubMed

    Kjaergaard, Christian H; Qayyum, Munzarin F; Wong, Shaun D; Xu, Feng; Hemsworth, Glyn R; Walton, Daniel J; Young, Nigel A; Davies, Gideon J; Walton, Paul H; Johansen, Katja Salomon; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I

    2014-06-17

    Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy. PMID:24889637

  1. 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

  2. Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

    PubMed Central

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

    2015-01-01

    Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace. PMID:25616801

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

    PubMed

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

    2015-04-01

    Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace. PMID:25616801

  4. The chemistry of aminoguanidine derivatives - preparation, crystal structure, thermal properties, and molecular docking studies of aminoguanidinium salts of several carboxylic acids

    NASA Astrophysics Data System (ADS)

    Selvakumar, Rajendran; Geib, Steven J.; Muthu Sankar, Aathi; Premkumar, Thathan; Govindarajan, Subbaiah

    2015-11-01

    The reaction of aminoguanidine bicarbonate (Amg) with oxamic, oxalic, malonic and sulfoacetic acids yielded (AmgH)H2NOC-COO (1), OOC-CONHNHC(NH2)NH2 (2) (AmgH)HOOC-CH2-COO (3) and O3S-CH2-CONHNHC(NH2)NH2 (4), respectively. For the first time, we studied the salt-forming ability of aminoguanidine with several carboxylic acids, such as oxamic, oxalic, malonic and sulphoacetic acids. We also compared the structural and thermal properties of these salts. Oxamic and malonic acids form only mono-aminoguanidinium salts, whereas oxalic acid mainly forms di-aminoguanidinium oxalate. In addition, oxalic acid forms guanylhydrazido-oxalic acid which exists as zwitter ion. Unlike other acids, sulfoacetic acid readily forms only the zwitter ionic salts (2-guanylhydrazido-oxo-methanesulfonic acid) rather than the usual simple salt. This result may be a result of the highly acidic nature of the sulfonic group, which favors acid catalyzed condensation. More significantly, for the first time, the ability guanylhydrazido-oxalic acid (2) and 2-guanylhydrazido-oxo-methanesulfonic acid (4) to inhibit human butyrylcholinesterase (human BChE) receptor has been studied with a molecular docking approach. The binding of the compounds to human BChE was examined as it is crucial to understanding the biological significance of aminoguanidine derivatives. The compounds were identified and characterized by analytical, FT-IR spectroscopic and thermal studies. Furthermore, the structures of compounds 1, 2 and 4 were confirmed by single X-ray diffraction studies. Compounds 1 and 2 crystallized in a monoclinic crystal system with P21/c and Cc space groups, respectively, whereas compound 4 crystalized in an orthorhombic system with a Pbca space group. All the compounds (1-4) underwent endo- followed by exothermic decomposition in the temperature range from 130 to 600 °C to yield gaseous products.

  5. On the source of organic acid aerosol layers above clouds.

    PubMed

    Sorooshian, Armin; Lu, Miao-Ling; Brechtel, Fred J; Jonsson, Haflidi; Feingold, Graham; Flagan, Richard C; Seinfeld, John H

    2007-07-01

    During the July 2005 Marine Stratus/Stratocumulus Experiment (MASE) and the August-September 2006 Gulf of Mexico Atmospheric Composition and Climate Study (GoMACCS), the Center for Interdisciplinary Remotely-Piloted Aircraft Studies (CIRPAS) Twin Otter probed aerosols and cumulus clouds in the eastern Pacific Ocean off the coast of northern California and in southeastern Texas, respectively. An on-board particle-into-liquid sampler (PILS) quantified inorganic and organic acid species with < or = 5-min time resolution. Ubiquitous organic aerosol layers above cloud with enhanced organic acid levels were observed in both locations. The data suggest that aqueous-phase reactions to produce organic acids, mainly oxalic acid, followed by droplet evaporation is a source of elevated organic acid aerosol levels above cloud. Oxalic acid is observed to be produced more efficiently relative to sulfate as the cloud liquid water content increases, corresponding to larger and less acidic droplets. As derived from large eddy simulations of stratocumulus underthe conditions of MASE, both Lagrangian trajectory analysis and diurnal cloudtop evolution provide evidence that a significant fraction of the aerosol mass concentration above cloud can be accounted for by evaporated droplet residual particles. Methanesulfonate data suggest that entrainment of free tropospheric aerosol can also be a source of organic acids above boundary layer clouds. PMID:17695910

  6. Identification of a novel Baeyer‐Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA

    PubMed Central

    Minerdi, Daniela; Zgrablic, Ivan; Sadeghi, Sheila J.; Gilardi, Gianfranco

    2012-01-01

    Summary This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long‐chain alkanes as the sole energy source expresses almA gene coding for a Baeyer‐Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer‐Villiger reactions as well as oxidation of the prodrug ethionamide. PMID:22862894

  7. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

    DOE PAGESBeta

    Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; Keul, Nicholas D.; Rodriguez, Pedro J.; Robinson, Howard H.; Mattevi, Andrea; Sobrado, Pablo

    2015-03-23

    N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producingmore » more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.« less

  8. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

    SciTech Connect

    Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; Keul, Nicholas D.; Rodriguez, Pedro J.; Robinson, Howard H.; Mattevi, Andrea; Sobrado, Pablo

    2015-03-23

    N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.

  9. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation*

    PubMed Central

    Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; Keul, Nicholas D.; Rodriguez, Pedro J.; Robinson, Howard H.; Mattevi, Andrea; Sobrado, Pablo

    2015-01-01

    N-Hydroxylating monooxygenases are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on d-Lys, although it binds l-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA, and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. Biological implications of these findings are discussed. PMID:25802330

  10. Role of P450 Monooxygenases in the Degradation of the Endocrine-Disrupting Chemical Nonylphenol by the White Rot Fungus Phanerochaete chrysosporium▿

    PubMed Central

    Subramanian, Venkataramanan; Yadav, Jagjit S.

    2009-01-01

    The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (∼75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant. PMID:19542331

  11. Non-hazardous Baeyer-Villiger oxidation of levulinic acid derivatives: alternative renewable access to 3-hydroxypropionates.

    PubMed

    Fink, Michael J; Mihovilovic, Marko D

    2015-02-18

    Baeyer-Villiger monooxygenases catalyze the energetically challenging oxidation of levulinates (4-oxopentanoates) to 3-hydroxypropionic acid (3-HPA) derivates under ambient conditions, replacing propellant-grade H2O2 with aerial oxygen as the oxidant. This reaction enables a new pathway to a platform for chemical 3-HPA, an important intermediate in the non-petrol based production of a variety of bulk chemicals (acrylates, malonates, 1,3-propanediol). PMID:25583122

  12. Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein.

    PubMed

    Lountos, George T; Mitchell, Kevin H; Studts, Joey M; Fox, Brian G; Orville, Allen M

    2005-05-17

    Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complex is a 102-residue protein devoid of metals or organic cofactors. It forms a complex with the diiron hydroxylase component (T4moH) that influences both the kinetics and regiospecificity of catalysis. Here, we report crystal structures for native T4moD and two engineered variants with either four (DeltaN4-) or 10 (DeltaN10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-A resolution, respectively. The crystal structures have C-alpha root-mean-squared differences of less than 0.8 A for the central core consisting of residues 11-98, showing that alterations of the N-terminal have little influence on the folded core of the protein. The central core has the same fold topology as observed in the NMR structures of T4moD, the methane monooxygenase effector protein (MmoB) from two methanotrophs, and the phenol hydroxylase effector protein (DmpM). However, the root-mean-squared differences between comparable C-alpha positions in the X-ray structures and the NMR structures vary from approximately 1.8 A to greater than 6 A. The X-ray structures exhibit an estimated overall coordinate error from 0.095 (0.094) A based on the R-value (R free) for the highest resolution DeltaN4-T4moD structure to 0.211 (0.196) A for the native T4moD structure. Catalytic studies of the DeltaN4-, DeltaN7-, and DeltaN10- variants of T4moD show statistically insignificant changes in k(cat), K(M), k(cat)/K(M), and K(I) relative to the native protein. Moreover, there was no significant change in the regiospecificity of toluene oxidation with any of the T4moD variants. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the approximately 33 residue N-terminal region for catalytic activity. PMID:15882052

  13. 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

  14. Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism.

    PubMed

    Kim, Seonah; Ståhlberg, Jerry; Sandgren, Mats; Paton, Robert S; Beckham, Gregg T

    2014-01-01

    Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η(1)-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η(1)) to copper, and that a copper-oxyl-mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds. PMID:24344312

  15. Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina.

    PubMed

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis. PMID:23124232

  16. Tertiary amines related to brompheniramine: preferred conformations for N-oxygenation by the hog liver flavin-containing monooxygenase.

    PubMed

    Cashman, J R; Celestial, J R; Leach, A; Newdoll, J; Park, S B

    1993-08-01

    The metabolism of racemic, (D)- and (L)-brompheniramine, a widely used antihistamine, was studied with microsomes and with highly purified flavin-containing monooxygenase (FMO) from hog liver. In addition, a number of other similar tertiary amines were evaluated as substrates for FMO activity from hog liver and the kinetic constants obtained were compared with brompheniramine. Although some N-demethylation was observed, the major metabolite of brompheniramine and the other tertiary amines examined in hog liver microsomes was the metabolite containing an aliphatic nitrogen N-oxide. Brompheniramine was extensively N-oxygenated by the highly purified FMO from hog liver. N-Oxygenation of brompheniramine in both microsomes and with highly purified FMO from hog liver was enantioselective. The Km for N-oxygenation of (D)-brompheniramine was markedly lower than the Km for (L)-brompheniramine. (E)- and (Z)-zimeldine are less conformationally flexible model compounds of brompheniramine, and these compounds were also examined and were found to be stereoselectively N-oxygenated by the highly purified FMO from hog liver. The similarities and differences in Km and Vmax values were evaluated in terms of possible conformations of the substrates determined by SYBYL molecular mechanics calculations. Distance map data indicated that FMO preferentially accommodated selected conformations of tertiary amines. Thus, (D)-brompheniramine and (Z)-zimeldine presumably have the aliphatic tertiary amine nitrogen atom and aromatic ring center at a defined distance and geometry and were more efficiently N-oxygenated than their respective isomers. PMID:8415393

  17. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    PubMed Central

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    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

  18. Prognostic significance of kynurenine 3-monooxygenase and effects on proliferation, migration, and invasion of human hepatocellular carcinoma.

    PubMed

    Jin, Haojie; Zhang, Yurong; You, Haiyan; Tao, Xuemei; Wang, Cun; Jin, Guangzhi; Wang, Ning; Ruan, Haoyu; Gu, Dishui; Huo, Xisong; Cong, Wenming; Qin, Wenxin

    2015-01-01

    Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the kynurenine pathway of tryptophan degradation and plays a critical role in Huntington's and Alzheimer's diseases. This study aimed to examine the expression of KMO in human hepatocellular carcinoma (HCC) and investigate the relationship between its expression and prognosis of HCC patients. We first analyzed KMO expression in 120 paired HCC samples (HCC tissues vs matched adjacent non-cancerous liver tissues), and 205 clinical HCC specimens using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis showed that KMO expression was significantly higher in HCC tissues than that in normal liver tissues (all p < 0.05). Survival and recurrence analyses showed that KMO was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR) (both p<0.01). And in vitro studies revealed that KMO positively regulated proliferation, migration, and invasion of HCC cells. These results suggest that KMO exhibits tumor-promoting effects towards HCC and it may serve as a novel prognostic marker in HCC. PMID:26099564

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

    PubMed

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

    2013-07-01

    The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. PMID:23568529

  20. A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus.

    PubMed Central

    St-Pierre, B.; De Luca, V.

    1995-01-01

    Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus. Tabersonine 16-hydroxylase (16-OH) activity was detected in total protein extracts from young leaves of C. roseus using a novel coupled assay system. Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole, and cytochrome c. 16-OH was localized to the endoplasmic reticulum by linear sucrose density gradient centrifugation. These data suggest that 16-OH is a cytochrome P-450-dependent monooxygenase. The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was induced by light. The leaf-specific distribution of 16-OH in the mature plant is consistent with the localization of other enzymes in the tabersonine to vindoline pathway. However, in contrast to enzymes that catalyze the last four steps of vindoline biosynthesis, enzymes responsible for the first two steps from tabersonine (16-OH and 16-O-methyltransfersase) were detected in C. roseus cell-suspension cultures. These data complement the complex model of vindoline biosynthesis that has evolved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms. PMID:12228585

  1. Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker ( sup 3 H)GBR-12935

    SciTech Connect

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

    1990-02-26

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

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

    PubMed Central

    Ismail, Alexandre; Leroux, Vincent; Smadja, Myriam; Gonzalez, Lucie; Lombard, Murielle; Pierrel, Fabien; Mellot-Draznieks, Caroline; Fontecave, Marc

    2016-01-01

    Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis. PMID:26808124

  3. Local Auxin Biosynthesis Mediated by a YUCCA Flavin Monooxygenase Regulates Haustorium Development in the Parasitic Plant Phtheirospermum japonicum.

    PubMed

    Ishida, Juliane K; Wakatake, Takanori; Yoshida, Satoko; Takebayashi, Yumiko; Kasahara, Hiroyuki; Wafula, Eric; dePamphilis, Claude W; Namba, Shigetou; Shirasu, Ken

    2016-08-01

    Parasitic plants in the Orobanchaceae cause serious agricultural problems worldwide. Parasitic plants develop a multicellular infectious organ called a haustorium after recognition of host-released signals. To understand the molecular events associated with host signal perception and haustorium development, we identified differentially regulated genes expressed during early haustorium development in the facultative parasite Phtheirospermum japonicum using a de novo assembled transcriptome and a customized microarray. Among the genes that were