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Sample records for key active site

  1. Key Role of Active-Site Water Molecules in Bacteriorhodopsin Proton-Transfer Reactions

    SciTech Connect

    Bondar, A.N.; Baudry, Jerome Y; Suhai, Sandor; Fischer, S.; Smith, Jeremy C

    2008-10-01

    The functional mechanism of the light-driven proton pump protein bacteriorhodopsin depends on the location of water molecules in the active site at various stages of the photocycle and on their roles in the proton-transfer steps. Here, free energy computations indicate that electrostatic interactions favor the presence of a cytoplasmic-side water molecule hydrogen bonding to the retinal Schiff base in the state preceding proton transfer from the retinal Schiff base to Asp85. However, the nonequilibrium nature of the pumping process means that the probability of occupancy of a water molecule in a given site depends both on the free energies of insertion of the water molecule in this and other sites during the preceding photocycle steps and on the kinetic accessibility of these sites on the time scale of the reaction steps. The presence of the cytoplasmic-side water molecule has a dramatic effect on the mechanism of proton transfer: the proton is channeled on the Thr89 side of the retinal, whereas the transfer on the Asp212 side is hindered. Reaction-path simulations and molecular dynamics simulations indicate that the presence of the cytoplasmic-side water molecule permits a low-energy bacteriorhodopsin conformer in which the water molecule bridges the twisted retinal Schiff base and the proton acceptor Asp85. From this low-energy conformer, proton transfer occurs via a concerted mechanism in which the water molecule participates as an intermediate proton carrier.

  2. Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity.

    PubMed

    Talens-Perales, David; Polaina, Julio; Marín-Navarro, Julia

    2016-04-13

    Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3'-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3'-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold. PMID:26998654

  3. Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

    PubMed

    Schoch, Guillaume A; Attias, Roger; Le Ret, Monique; Werck-Reichhart, Danièle

    2003-09-01

    CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation. PMID:12950252

  4. Protonation states of the key active site residues and structural dynamics of glmS riboswitch as reveled by molecular dynamics

    PubMed Central

    Banáš, Pavel; Walter, Nils G.

    2010-01-01

    The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous Gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, detailed reaction mechanism as well as protonation state of glmS reactive form remains still elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the T. tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40− seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase of Km with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. Based on the identified dominant protonation state of the reaction precursor, we propose a hypothesis of self-cleavage mechanism, in which A-1(2'-OH) is activated as nucleophile by the G1(pro-Rp) non-bridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group. PMID:20536206

  5. Probing the Active Site of MIO-dependent Aminomutases, Key Catalysts in the Biosynthesis of amino Acids Incorporated in Secondary Metabolites

    SciTech Connect

    Cooke, H.; Bruner, S

    2010-01-01

    The tyrosine aminomutase SgTAM produces (S)-{beta}-tyrosine from L-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form {alpha},{beta}-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been limited reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the {alpha},{beta}-unsaturated intermediates to form {beta}-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis.

  6. Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of beta-amino acids incorporated in secondary metabolites.

    PubMed

    Cooke, Heather A; Bruner, Steven D

    2010-09-01

    The tyrosine aminomutase SgTAM produces (S)-ss-tyrosine from L-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form alpha,ss-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been limited reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the alpha,ss-unsaturated intermediates to form ss-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis. PMID:20577998

  7. Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

    SciTech Connect

    Fang, Ti; Li, De-Feng; Zhou, Ning-Yi

    2011-07-08

    Highlights: {yields} Application of site-directed mutagenesis to probe the active site residues of glutathione-dependent maleylpyruvate isomerase. {yields} Two conserved residues, Arg8 and Arg176, in zeta class glutathione S-transferases are critical for maleylpyruvate orientation and enolization. {yields} Arg109, found exclusively in NagL, participates in k{sub cat} regulation. {yields} The T11A mutant exhibited a significantly decreased K{sub m} value for glutathione with little impact on maleylpyruvate kinetics. {yields} The Thr11 residue appears to have significance in the evolution of glutathione S-transferase classes. -- Abstract: The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k{sub cat} regulation. Surprisingly, the T11A mutant had a

  8. Keys To Protecting Your Observing Site

    NASA Astrophysics Data System (ADS)

    Hanslep, D. A.; Crawford, D. L.

    2005-05-01

    Everyone knows that astronomers need dark skies to be able to work. However, with the rapid spread of development across the entire USA, the sky brightness background has been increasing in most areas at an alarming rate. Recent nighttime satellite photography shows once pristine observing sites now being threatened by the increased light pollution that accompanies urban sprawl. But while few observatories have programs to combat this plague on astronomy, the International Dark-Sky Association has instituted a number of programs to protect this natural, cultural resource - and they've been succeeding. In working effectively with the professional lighting community, local authorities, and elected officials to produce results in dark sky preservation through outdoor lighting ordinances, public education, technical committee service and alliance development, IDA has found the formula for extending the lifespan of today's observing sites, and needs astronomy's support. The International Dark-Sky Association is a membership-based 501(c)(3) non-profit organization dedicated to protecting and restoring the natural night environment and our heritage of dark skies through the promotion of quality outdoor lighting.

  9. Floor Plan, Axonometric View, Site Location Key, Cesar Chavez Fasting ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Floor Plan, Axonometric View, Site Location Key, Cesar Chavez Fasting Room Diagram - Forty Acres, Tomasa Zapata Mireles Co-op Building , 30168 Garces Highway (Northwest Corner of Garces Highway and Mettler Avenue), Delano, Kern County, CA

  10. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    PubMed

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL. PMID:26820485

  11. 21. Photographic copy of a keyed site plan for North ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    21. Photographic copy of a keyed site plan for North Base: Military Construction, Muroc Flight Test Base, Muroc, California; General Layout Plan, December 1944. Reproduced from the holdings of the National Archives, Pacific Southwest Region - Edwards Air Force Base, North Base, North Base Road, Boron, Kern County, CA

  12. 75 FR 20364 - Public Buildings Service; Key Largo Beacon Annex Site; Key Largo, FL; Transfer of Property

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-19

    ... From the Federal Register Online via the Government Publishing Office GENERAL SERVICES ADMINISTRATION Public Buildings Service; Key Largo Beacon Annex Site; Key Largo, FL; Transfer of Property..., identified as Key Largo Beacon Annex Site, Key Largo, FL to the U.S. Fish and Wildlife Service, Department...

  13. Identification and determination of trapping parameters as key site parameters for CO2 storage for the active CO2 storage site in Ketzin (Germany) - Comparison of different experimental approaches and analysis of field data

    NASA Astrophysics Data System (ADS)

    Zemke, Kornelia; Liebscher, Axel

    2015-04-01

    Petrophysical properties like porosity and permeability are key parameters for a safe long-term storage of CO2 but also for the injection operation itself. The accurate quantification of residual trapping is difficult, but very important for both storage containment security and storage capacity; it is also an important parameter for dynamic simulation. The German CO2 pilot storage in Ketzin is a Triassic saline aquifer with initial conditions of the target sandstone horizon of 33.5 ° C/6.1 MPa at 630 m. One injection and two observation wells were drilled in 2007 and nearly 200 m of core material was recovered for site characterization. From June 2008 to September 2013, slightly more than 67 kt food-grade CO2 has been injected and continuously monitored. A fourth observation well has been drilled after 61 kt injected CO2 in summer 2012 at only 25 m distance to the injection well and new core material was recovered that allow study CO2 induced changes in petrophysical properties. The observed only minor differences between pre-injection and post-injection petrophysical parameters of the heterogeneous formation have no severe consequences on reservoir and cap rock integrity or on the injection behavior. Residual brine saturation for the Ketzin reservoir core material was estimated by different methods. Brine-CO2 flooding experiments for two reservoir samples resulted in 36% and 55% residual brine saturation (Kiessling, 2011). Centrifuge capillary pressure measurements (pc = 0.22 MPa) yielded the smallest residual brine saturation values with ~20% for the lower part of the reservoir sandstone and ~28% for the upper part (Fleury, 2010). The method by Cerepi (2002), which calculates the residual mercury saturation after pressure release on the imbibition path as trapped porosity and the retracted mercury volume as free porosity, yielded unrealistic low free porosity values of only a few percent, because over 80% of the penetrated mercury remained in the samples after

  14. Key site abandonment steps in CO2 storage

    NASA Astrophysics Data System (ADS)

    Kühn, M.; Wipki, M.; Durucan, S.; Deflandre, J.-P.; Lüth, S.; Wollenweber, J.; Chadwick, A.; Böhm, G.

    2012-04-01

    CO2CARE is an EU funded project within FP7-research, which started in January 2011 with a funding period of three years. The project objectives will be achieved through an international consortium consisting of 23 partners from Europe, USA, Canada, Japan, and Australia, belonging to universities, research institutes, and energy companies. According to the EC Guidance Document 3, the lifetime of a CO2 storage site can be generally subdivided into 6 phases: 1. assessment, 2. characterisation, 3. development, 4. operation, 5. post-closure/pre-transfer, and 6. post transfer. CO2CARE deals with phases 5 and 6. The main goals of the project are closely linked to the three high-level requirements of the EU Directive 2009/31/EC, Article 18 for CO2 storage which are: (i) absence of any detectable leakage, (ii) conformity of actual behaviour of the injected CO2 with the modelled behaviour, and (iii) the storage site is evolving towards a situation of long-term stability. These criteria have to be fulfilled prior to subsequent transfer of responsibility to the competent authorities, typically 20 or 30 years after site closure. CO2CARE aims to formulate robust procedures for site abandonment which will meet the regulatory requirements and ensure long-term integrity of the storage complex. We present key results from the first year of the project via a report on international regulatory requirements on CO2 geological storage and site abandonment that includes a general overview on the current state-of-the art in abandonment methodologies in the oil and gas industry worldwide. Due to the long time-frames involved in CO2 storage (in the range of several thousands of years), the behaviour of a system with respect to, for example, long-term well stability can be demonstrated only by using long-term predictive modelling tools to study potential leakage pathways. Trapping mechanisms for CO2 are of high interest concerning a quantitative estimation of physically captured, capillary

  15. SITE TECHNOLOGY CAPSULE: GIS\\KEY ENVIRONMENTAL DATA MANAGEMENT SYSTEM

    EPA Science Inventory

    GIS/Key™ is a comprehensive environmental database management system that integrates site data and graphics, enabling the user to create geologic cross-sections; boring logs; potentiometric, isopleth, and structure maps; summary tables; and hydrographs. GIS/Key™ is menu-driven an...

  16. Prominences: The Key to Understanding Solar Activity

    NASA Technical Reports Server (NTRS)

    Karpen, Judy T.

    2011-01-01

    Prominences are spectacular manifestations of both quiescent and eruptive solar activity. The largest examples can be seen with the naked eye during eclipses, making prominences among the first solar features to be described and catalogued. Steady improvements in temporal and spatial resolution from both ground- and space-based instruments have led us to recognize how complex and dynamic these majestic structures really are. Their distinguishing characteristics - cool knots and threads suspended in the hot corona, alignment along inversion lines in the photospheric magnetic field within highly sheared filament channels, and a tendency to disappear through eruption - offer vital clues as to their origin and dynamic evolution. Interpreting these clues has proven to be contentious, however, leading to fundamentally different models that address the basic questions: What is the magnetic structure supporting prominences, and how does so much cool, dense plasma appear in the corona? Despite centuries of increasingly detailed observations, the magnetic and plasma structures in prominences are poorly known. Routine measurements of the vector magnetic field in and around prominences have become possible only recently, while long-term monitoring of the underlying filament-channel formation process also remains scarce. The process responsible for prominence mass is equally difficult to establish, although we have long known that the chromosphere is the only plausible source. As I will discuss, however, the motions and locations of prominence material can be used to trace the coronal field, thus defining the magnetic origins of solar eruptions. A combination of observations, theory, and numerical modeling must be used to determine whether any of the competing theories accurately represents the physics of prominences. I will discuss the criteria for a successful prominence model, compare the leading models, and present in detail one promising, comprehensive scenario for

  17. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  18. Selectivity for strand-transfer over 3'-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme-DNA interactions in the active site.

    PubMed

    Métifiot, Mathieu; Johnson, Barry C; Kiselev, Evgeny; Marler, Laura; Zhao, Xue Zhi; Burke, Terrence R; Marchand, Christophe; Hughes, Stephen H; Pommier, Yves

    2016-08-19

    Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed. PMID:27369381

  19. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  20. Acid potentiation of the capsaicin receptor determined by a key extracellular site.

    PubMed

    Jordt, S E; Tominaga, M; Julius, D

    2000-07-01

    The capsaicin (vanilloid) receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. The response of VR1 to capsaicin or noxious heat is dynamically potentiated by extracellular protons within a pH range encountered during tissue acidosis, such as that associated with arthritis, infarction, tumor growth, and other forms of injury. A molecular determinant for this important physiological activity was localized to an extracellular Glu residue (E600) in the region linking the fifth transmembrane domain with the putative pore-forming region of the channel. We suggest that this residue serves as a key regulatory site of the receptor by setting sensitivity to other noxious stimuli in response to changes in extracellular proton concentration. We also demonstrate that protons, vanilloids, and heat promote channel opening through distinct pathways, because mutations at a second site (E648) selectively abrogate proton-evoked channel activation without diminishing responses to other noxious stimuli. Our findings provide molecular evidence for stimulus-specific steps in VR1 activation and offer strategies for the development of novel analgesic agents. PMID:10859346

  1. One Key LOGO and Hands-On Activity Cards.

    ERIC Educational Resources Information Center

    Friesen, Chuck; And Others

    Developed to assist primary school teachers who wish to implement LOGO and One-Key LOGO (OKL) in their schools, this document consists of a LOGO resource manual and 92 color-coded activity cards designed to guide a pre-reader or primary child through a series of problem solving steps. After a brief introduction, which contains computer terminology…

  2. Exploring Key Sustainable Development Themes through Learning Activities

    ERIC Educational Resources Information Center

    Cruickshank, Heather; Fenner, Richard

    2012-01-01

    Purpose: The purpose of the paper is to examine how a number of key themes are introduced in the Master's programme in Engineering for Sustainable Development, at Cambridge University, through student-centred activities. These themes include dealing with complexity, uncertainty, change, other disciplines, people, environmental limits, whole life…

  3. Active magnetic radiation shielding system analysis and key technologies

    NASA Astrophysics Data System (ADS)

    Washburn, S. A.; Blattnig, S. R.; Singleterry, R. C.; Westover, S. C.

    2015-01-01

    Many active magnetic shielding designs have been proposed in order to reduce the radiation exposure received by astronauts on long duration, deep space missions. While these designs are promising, they pose significant engineering challenges. This work presents a survey of the major systems required for such unconfined magnetic field design, allowing the identification of key technologies for future development. Basic mass calculations are developed for each system and are used to determine the resulting galactic cosmic radiation exposure for a generic solenoid design, using a range of magnetic field strength and thickness values, allowing some of the basic characteristics of such a design to be observed. This study focuses on a solenoid shaped, active magnetic shield design; however, many of the principles discussed are applicable regardless of the exact design configuration, particularly the key technologies cited.

  4. Active magnetic radiation shielding system analysis and key technologies.

    PubMed

    Washburn, S A; Blattnig, S R; Singleterry, R C; Westover, S C

    2015-01-01

    Many active magnetic shielding designs have been proposed in order to reduce the radiation exposure received by astronauts on long duration, deep space missions. While these designs are promising, they pose significant engineering challenges. This work presents a survey of the major systems required for such unconfined magnetic field design, allowing the identification of key technologies for future development. Basic mass calculations are developed for each system and are used to determine the resulting galactic cosmic radiation exposure for a generic solenoid design, using a range of magnetic field strength and thickness values, allowing some of the basic characteristics of such a design to be observed. This study focuses on a solenoid shaped, active magnetic shield design; however, many of the principles discussed are applicable regardless of the exact design configuration, particularly the key technologies cited. PMID:26177618

  5. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  6. A QUICK KEY TO THE SUBFAMILIES AND GENERA OF ANTS OF THE SAVANNAH RIVER SITE

    SciTech Connect

    Martin, D

    2007-09-04

    This taxonomic key was devised to support development of a Rapid Bioassessment Protocol using ants at the Savannah River Site. The emphasis is on 'rapid' and, because the available keys contained a very large number of genera not known to occur at the Savannah River Site, we found that the available keys were unwieldy. Because these keys contained many more genera than we would ever encounter and because this larger number of genera required more couplets in the key and often required examination of characters that are difficult to assess without higher magnifications (60X or higher), more time was required to process samples. In developing this set of keys I emphasized character states that are easier for nonspecialists to recognize. I recognize that the character sets used may lead to some errors but I believe that the error rate will be small and, for the purpose of rapid bioassessment, this error rate will be acceptable provided that overall sample sizes are adequate. Oliver and Beattie (1996a, 1996b) found that for rapid assessment of biodiversity the same results were found when identifications were done to morphospecies by people with minimal expertise as when the same data sets were identified by subject matter experts. Basset et al. (2004) concluded that it was not as important to correctly identify all species as it was to be sure that the study included as many functional groups as possible. If your study requires high levels of accuracy, it is highly recommended that, when you key out a specimen and have any doubts concerning the identification, you should refer to keys in Bolton (1994) or to the other keys used to develop this area specific taxonomic key.

  7. Key residues at the riboflavin kinase catalytic site of the bifunctional riboflavin kinase/FMN adenylyltransferase from Corynebacterium ammoniagenes.

    PubMed

    Serrano, Ana; Frago, Susana; Herguedas, Beatriz; Martínez-Júlvez, Marta; Velázquez-Campoy, Adrián; Medina, Milagros

    2013-01-01

    Many known prokaryotic organisms depend on a single bifunctional enzyme, encoded by the RibC of RibF gene and named FAD synthetase (FADS), to convert Riboflavin (RF), first into FMN and then into FAD. The reaction occurs through the sequential action of two activities present on a single polypeptide chain where the N-terminus is responsible for the ATP:FMN adenylyltransferase (FMNAT) activity and the C-terminus for the ATP: riboflavin kinase (RFK) activity. Sequence and structural analysis suggest that T208, N210 and E268 at the C-terminus RFK module of Corynebacterium ammoniagenes FADS (CaFADS) might be key during RF phosphorylation. The effect of site-directed mutagenesis on the RFK activity, as well as on substrates and products binding, indicates that T208 and N210 provide the RFK active-site geometry for binding and catalysis, while E268 might be involved in the catalytic step as catalytic base. These data additionally suggest concerted conformational changes at the RFK module of CaFADS during its activity. Mutations at the RFK site also modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD. This observation supports the hypothesis that the hexameric assembly previously revealed by the crystal structure of CaFADS might play a functional role during catalysis. PMID:22892871

  8. Key gravity-sensitive signaling pathways drive T cell activation.

    PubMed

    Boonyaratanakornkit, J B; Cogoli, A; Li, C-F; Schopper, T; Pippia, P; Galleri, G; Meloni, M A; Hughes-Fulford, M

    2005-12-01

    Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity. PMID:16210397

  9. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  10. KEY ELEMENTS OF CHARACTERIZING SAVANNAH RIVER SITE HIGH LEVEL WASTE SLUDGE INSOLUBLES THROUGH SAMPLING AND ANALYSIS

    SciTech Connect

    Reboul, S; Barbara Hamm, B

    2007-05-24

    Characterization of HLW is a prerequisite for effective planning of HLW disposition and site closure performance assessment activities. Adequate characterization typically requires application of a combination of data sources, including process knowledge, theoretical relationships, and real-waste analytical data. Consistently obtaining high quality real-waste analytical data is a challenge, particularly for HLW sludge insolubles, due to the inherent complexities associated with matrix heterogeneities, sampling access limitations, radiological constraints, analyte loss mechanisms, and analyte measurement interferences. Understanding how each of these complexities affects the analytical results is the first step to developing a sampling and analysis program that provides characterization data that are both meaningful and adequate. A summary of the key elements impacting SRS HLW sludge analytical data uncertainties is presented in this paper, along with guidelines for managing each of the impacts. The particular elements addressed include: (a) sample representativeness; (b) solid/liquid phase quantification effectiveness; (c) solids dissolution effectiveness; (d) analyte cross contamination, loss, and tracking; (e) dilution requirements; (f) interference removal; (g) analyte measurement technique; and (h) analytical detection limit constraints. A primary goal of understanding these elements is to provide a basis for quantifying total propagated data uncertainty.

  11. Analysis of landslide overgrowing rates at Vaskiny Dachi key site, Central Yamal, Russia

    NASA Astrophysics Data System (ADS)

    Khomutov, A.

    2009-04-01

    An estimation of overgrowing of landslide-affected slopes by vegetation at three main landslide elements: shear surface, landslide body and "frontal zone" at Vaskiny Dachi key site is presented. Vaskiny Dachi key site is located in the watershed of Se-Yakha and Mordy-Yakha rivers on Central Yamal, Russia. The area is represented by highly-dissected alluvial-lacustrine-marine plains and terraces. The closest to Vaskiny Dachi climate station is Marresale, about 90 km southwest of Vaskiny Dachi, at the Kara sea coast. The weather here is probably somewhat cooler than at Vaskiny Dachi. The average annual (summer) air temperature at Marresale is -8.3° C (4.3° C) ("Russia's Weather" Server). To estimate vegetation cover dynamics on cryogenic landslides at "Vaskiny Dachi", data published by O.Rebristaya and others (1995) were used. Their observations were done in 1991-1993, and were supplemented by further field observations (Leibman et al., 2000, Khomutov & Leibman 2007) and by field and remote sensing observations in 2008. An estimation of vegetation cover dynamics on cryogenic landslides at "Vaskiny Dachi" leads to the following results. Immediately after landsliding in 1989, landslide shear surface was bare without any vegetation, landslide body had initial vegetation, and "frontal zone" was under liquefied sediment masses. "Frontal zone" formed in front of a landslide body, appears as a result of damming of drainage routes by a landslide body with flooding of the shear surface "upstream" of the landslide body, formation of a sedge-cottongrass meadow there, and swamping downstream (Khomutov & Leibman 2007). By 1993, landslide shear surface got overgrown by species subordinate in surrounding initial landscapes (Alopecurus alpinus, Festuca ovina, Calamagrostis neglecta, Poa alpigena ssp. Alpigena, etc.). Landslide body was covered by initial communities which got depressed: vitality of Salix polaris, Vaccinium vitis-idaea was reduced, dead off moss cover and overgrown

  12. Active listening: The key of successful communication in hospital managers

    PubMed Central

    Jahromi, Vahid Kohpeima; Tabatabaee, Seyed Saeed; Abdar, Zahra Esmaeili; Rajabi, Mahboobeh

    2016-01-01

    Introduction One of the important causes of medical errors and unintentional harm to patients is ineffective communication. The important part of this skill, in case it has been forgotten, is listening. The objective of this study was to determine whether managers in hospitals listen actively. Methods This study was conducted between May and June 2014 among three levels of managers at teaching hospitals in Kerman, Iran. Active Listening skill among hospital managers was measured by self-made Active Listening Skill Scale (ALSS), which consists of the key elements of active listening and has five subscales, i.e., Avoiding Interruption, Maintaining Interest, Postponing Evaluation, Organizing Information, and Showing Interest. The data were analyzed by IBM-SPSS software, version 20, and the Pearson product-moment correlation coefficient, the chi-squared test, and multiple linear regressions. Results The mean score of active listening in hospital managers was 2.32 out of 3.The highest score (2.27) was obtained by the first-level managers, and the top managers got the lowest score (2.16). Hospital mangers were best in showing interest and worst in avoiding interruptions. The area of employment was a significant predictor of avoiding interruption and the managers’ gender was a strong predictor of skill in maintaining interest (p < 0.05). The type of management and education can predict postponing evaluation, and the length of employment can predict showing interest (p < 0.05). Conclusion There is a necessity for the development of strategies to create more awareness among the hospital managers concerning their active listening skills. PMID:27123221

  13. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  14. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  15. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  16. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  17. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  18. Floridas Miami Tequesta Indian Site, Its Calusa Indian Locations, the Matacumbe Keys, and Orlandos Wikiwa Springs Generate Environmentally Significant EMFs.

    NASA Astrophysics Data System (ADS)

    Mac Dougall, Jean S.; Mc Leod, Roger D.; Mc Leod, David M.

    2003-10-01

    Florida purchased the Tequesta ([Langue] doc Christ Spirit-signal) Indian site along the Miami River site that vigorously pulsates with even minor rainstorms entering or leaving the area. Although there is a laughable chimera of a fountain of youth associated with Ponce de Leons discovery of the Florida peninsula in about AD 1513, the Calusa (Royal Christ Jesus Spirit-signal) Indian Nation has an associated significance with EMF signals they possibly monitored throughout their area of activity. Our efforts have also led to the investigation of cultural and other influences implied by the Matacumbe Keys that indicate a shared commonality of awareness with Native Americans of the northeast such as Metacomet, or regions like Maines Grand Lake Matagamon and its associated electromagnetic Spirit Signal. Wikiwa Springs near Orlando shares much with Massachusetts (adherent of serpent Jesus Christ Spirit-signal) Natick, and New Hampshires Naticook Island. These are the locales of environmentally sensitive instrumentation.

  19. The SEDIBUD (Sediment Budgets in Cold Environments) Programme: Current activities and future key tasks

    NASA Astrophysics Data System (ADS)

    Beylich, A. A.; Lamoureux, S. F.; Decaulne, A.

    2012-04-01

    Projected climate change in cold regions is expected to alter melt season duration and intensity, along with the number of extreme rainfall events, total annual precipitation and the balance between snowfall and rainfall. Similarly, changes to the thermal balance are expected to reduce the extent of permafrost and seasonal ground frost and increase active layer depths. These effects will undoubtedly change surface environments in cold regions and alter the fluxes of sediments, nutrients and solutes, but the absence of quantitative data and coordinated process monitoring and analysis to understand the sensitivity of the Earth surface environment is acute in cold climate environments. The International Association of Geomorphologists (I.A.G./A.I.G.)SEDIBUD (Sediment Budgets in Cold Environments) Programme was formed in 2005 to address this existing key knowledge gap. SEDIBUD currently has about 400 members worldwide and the Steering Committee of this international programme is composed of ten scientists from eight different countries: Achim A. Beylich (Chair) (Norway), Armelle Decaulne (Secretary) (France), John C. Dixon (USA), Scott F. Lamoureux (Vice-Chair) (Canada), John F. Orwin (Canada), Jan-Christoph Otto (Austria), Irina Overeem (USA), Thorsteinn Saemundsson (Iceland), Jeff Warburton (UK), Zbigniew Zwolinski (Poland). The central research question of this global group of scientists is to: Assess and model the contemporary sedimentary fluxes in cold climates, with emphasis on both particulate and dissolved components. Initially formed as European Science Foundation (ESF) Network SEDIFLUX (2004-2006), SEDIBUD has further expanded to a global group of researchers with field research sites located in polar and alpine regions in the northern and southern hemisphere. Research carried out at each of the close to 50 defined SEDIBUD key test sites varies by programme, logistics and available resources, but typically represent interdisciplinary collaborations of

  20. Increase in physical activities in kindergarten children with cerebral palsy by employing MaKey-MaKey-based task systems.

    PubMed

    Lin, Chien-Yu; Chang, Yu-Ming

    2014-09-01

    In this study, we employed Flash- and Scratch-based multimedia by using a MaKey-MaKey-based task system to increase the motivation level of children with cerebral palsy to perform physical activities. MaKey MaKey is a circuit board that converts physical touch to a digital signal, which is interpreted by a computer as a keyboard message. In this study, we used conductive materials to control this interaction. This study followed single-case design using ABAB models in which A indicated the baseline and B indicated the intervention. The experiment period comprised 1 month and a half. The experimental results demonstrated that in the case of two kindergarten children with cerebral palsy, their scores were considerably increased during the intervention phrases. The developmental applications of the results are also discussed. PMID:24864049

  1. The Amazon tall tower observatory (ATTO) site - Multi-year aerosol observations and scientific key questions

    NASA Astrophysics Data System (ADS)

    Pöhlker, C.; Barbosa, H. M.; Brito, J.; Carbone, S.; Chi, X.; Kesselmeier, J.; Ditas, F.; Pöhlker, M. L.; Manzi, A. O.; Moran, D.; Poeschl, U.; Ruckteschler, N.; Saturno, J.; Soergel, M.; Su, H.; Walter, D.; Wang, Q.; Wang, Z.; Weber, B.; Wolff, S.; Yanez-Serrano, A. M.; Artaxo, P.; Andreae, M. O.

    2015-12-01

    The Amazon tall tower observatory site is located 150 km NE of Manaus in undisturbed rain forest areas. It serves as a remote measurement station in the Amazon forest with continuous aerosol, trace gas, micrometeorological, and ecological measurements. During part of the rainy season, the atmospheric state approximates pre-industrial conditions, in strong contrast to the dry season, which is dominated by significant pollution from deforestation fires and urban emissions. This presentation will focus on aerosol studies of the past three years. It aims to provide a brief overview of the characteristic seasonality of the aerosol burden at the ATTO site. Moreover, it will discuss the following key questions and current results of the ongoing observations: (i) During the wet season and in the absence of long-range advection of African aerosols, atmospheric conditions at the ATTO site approximate a pristine state, which reveals the genuine contribution of biogenic aerosols. Biogenic particles in the super- and submicron range have been observed and their properties as well as potential sources will be discussed. (ii) In contrast to the classical new particle formation, the occurrence of ultrafine particles is comparably sparse and mainly occurs as short 'bursts', indicating a rather localized character. Our current understanding of this phenomenon and its significance for the overall aerosol burden will be addressed. (iii) Aerosol absorptivity is mainly caused by black carbon, however, indications for the presence of other light absorbing aerosol species have been found. Current results on light absorbing aerosol under clean and polluted conditions will be presented. (iv) Aerosol particles at the ATTO site are typically strongly aged and comprise pronounced internal mixtures, with important implications for their properties. Microspectroscopic analysis helps to obtain insights into atmospheric processing and its impact on particle morphology and phase state.

  2. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  3. Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA.

    PubMed

    Julien, Jean-Philippe; Lee, Peter S; Wilson, Ian A

    2012-11-01

    Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals. PMID:23046130

  4. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  5. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  6. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer.

    PubMed

    Dinpajooh, Mohammadhasan; Martin, Daniel R; Matyushov, Dmitry V

    2016-01-01

    Enzymes in biology's energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  7. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    NASA Astrophysics Data System (ADS)

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  8. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  9. CONSIDERATION OF REACTION INTERMEDIATES IN STRUCTURE-ACTIVITY RELATIONSHIPS: A KEY TO UNDERSTANDING AND PREDICTION

    EPA Science Inventory

    Consideration of Reaction Intermediates in Structure- Activity Relationships: A Key to Understanding and Prediction

    A structure-activity relationship (SAR) represents an empirical means for generalizing chemical information relative to biological activity, and is frequent...

  10. Rapid movement of wastewater from on-site disposal systems into surface waters in the lower Florida Keys

    USGS Publications Warehouse

    Paul, John H.; McLaughlin, Molly R.; Griffin, Dale W.; Lipp, Erin K.; Stokes, Rodger; Rose, Joan B.

    2000-01-01

    Viral tracer studies have been used previously to study the potential for wastewater contamination of surface marine waters in the Upper and Middle Florida Keys. Two bacteriophages, the marine bacteriophage φHSIC and the Salmonella phage PRD1, were used as tracers in injection well and septic tank studies in Saddlebunch Keys of the Lower Florida Keys and in septic tank studies in Boot Key Harbor, Marathon, of the Middle Keys. In Boot Key Harbor, both phages were detected in a canal adjacent to the seeded septic tank within 3 h 15 min of the end of the seed period. The tracer was then detected at all sampling sites in Boot Key Harbor, including one on the opposite side of U. S. Highway 1 in Florida Bay, and at an Atlantic Ocean beach outside Boot Key Harbor. Rates of migration based on first appearance of the phage ranged from 1.7 to 57.5 m h-1. In Saddlebunch Keys, φHSIC and PRD1 were used to seed a residential septic tank and a commercial injection well. The septic tank tracer was not found in any surface water samples. The injection well tracer was first detected at a site most distant from the seed site, a channel that connected Sugarloaf Sound with the Atlantic Ocean. The rate of tracer migration from the injection well to this channel ranged from 66.8 to 141 m h-1. Both tracer studies showed a rapid movement of wastewater from on-site sewage treatment and disposal systems in a southeasterly direction toward the reef tract and Atlantic Ocean, with preferential movement through tidal channels. These studies indicate that wastewater disposal systems currently in widespread use in the Florida Keys can rapidly contaminate the marine environment.

  11. Key role of tissue plasminogen activator in neurovascular coupling

    PubMed Central

    Park, Laibaik; Gallo, Eduardo F.; Anrather, Josef; Wang, Gang; Norris, Erin H.; Paul, Justin; Strickland, Sidney; Iadecola, Costantino

    2008-01-01

    The increase in blood flow evoked by synaptic activity is essential for normal brain function and underlies functional brain imaging signals. Nitric oxide, a vasodilator released by NMDA receptor activation, is critical for the flow increase, but the factors linking NMDA receptor activity to nitric oxide-dependent hyperemia are poorly understood. Here, we show that tissue plasminogen activator (tPA), a serine protease implicated in NMDA receptor signaling, is required for the flow increase evoked by somatosensory stimulation. tPA acts by facilitating neuronal nitric oxide release, but this effect does not involve enhancement of NMDA currents or the associated intracellular Ca2+ rise. Rather, the evidence suggests that tPA controls NMDA-dependent nitric oxide synthesis by influencing the phosphorylation state of neuronal nitric oxide synthase. These findings unveil a previously unrecognized role of tPA in vital homeostatic mechanisms coupling NMDA receptor signaling with nitric oxide synthesis and local cerebral perfusion. PMID:18195371

  12. Key Residues Regulating the Reductase Activity of the Human Mitochondrial Apoptosis Inducing Factor.

    PubMed

    Villanueva, Raquel; Ferreira, Patricia; Marcuello, Carlos; Usón, Alejandro; Miramar, M Dolores; Peleato, M Luisa; Lostao, Anabel; Susin, Santos A; Medina, Milagros

    2015-08-25

    The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase. PMID:26237213

  13. Scholarly Use of the Web: What Are the Key Inducers of Links to Journal Web Sites?

    ERIC Educational Resources Information Center

    Vaughan, Liwen; Thelwall, Mike

    2003-01-01

    Reports on a study to investigate the factors that influence the creation of links to journal Web sites. Specific questions addressed are whether site age and site content are inducers of links to a journal's Web site as measured by the ratio of link counts to Journal Impact Factors. (Author/MES)

  14. Activeness as a key to counter democratic balance

    NASA Astrophysics Data System (ADS)

    Qian, Shen; Liu, Yijun; Galam, Serge

    2015-08-01

    According to the classic Galam model of opinion dynamics, each agent participates at each update of an opinion interaction. While the scheme gives everyone the same chance to influence others, in reality, social activity and influence vary considerably from one agent to another. To account for such a feature, we introduce a new individual attribute-"activeness"-which makes some agents more inclined than others at engaging in local discussions. To enhance the corresponding effect, opinion updates are shifted from all-out agent interaction cycles to few agent interaction cycles. Using dynamic analysis and simulations the resulting model is found to exhibit a "Minority Counteroffensive" phenomenon, which under some initial conditions makes the minority to win the opinion competition despite a threshold tipping point at fifty percent. The associated probabilistic phenomenon persists in the case "activeness" is held equal for all agents. The effect of "opinion leaders" is also investigated. Indeed, a leader is an inflexible agent, i.e., an agent who does not change opinion. The results reveal that two opinion leaders with moderate social influence may have a stronger effect than one opinion leader with a strong social influence. The model may shed a new light to the understanding of opinion formation and public voting.

  15. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  16. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  17. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  18. Keys to active ageing: new communication technologies and lifelong learning.

    PubMed

    Díaz-López, M Del Pilar; López-Liria, Remedios; Aguilar-Parra, José M; Padilla-Góngora, David

    2016-01-01

    The purpose of this study is to describe the creation and implementation of an ICT education program for the elderly in various Active Participation Centers in Almería (Spain), assessing its impact on quality of life. From a randomized sample of 200 individuals over the age of 55. Results reveal a high degree of participant satisfaction (76.6 %), as well as improvements in quality of life as compared to the control group after the 3 month program health factor: p = 0.004; leisure and activity factor: p = 0.001; Satisfaction with Life Factor: p < 0.001. The analysis conducted to determine the influence of age and gender on quality of life indicates that there are statistically significant differences in regards to age (the younger groups had higher scores) and gender (the males). This study may serve to facilitate similar works that promotes on-going education in different locations and across the lifespan. PMID:27386254

  19. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  20. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  1. 5 CFR 591.241 - What are the key activities of the COLA Advisory Committees?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false What are the key activities of the COLA Advisory Committees? 591.241 Section 591.241 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL... Areas Program Administration § 591.241 What are the key activities of the COLA Advisory Committees?...

  2. 5 CFR 591.241 - What are the key activities of the COLA Advisory Committees?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false What are the key activities of the COLA Advisory Committees? 591.241 Section 591.241 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL... Areas Program Administration § 591.241 What are the key activities of the COLA Advisory Committees?...

  3. 5 CFR 591.241 - What are the key activities of the COLA Advisory Committees?

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 1 2013-01-01 2013-01-01 false What are the key activities of the COLA Advisory Committees? 591.241 Section 591.241 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL... Areas Program Administration § 591.241 What are the key activities of the COLA Advisory Committees?...

  4. 5 CFR 591.241 - What are the key activities of the COLA Advisory Committees?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 5 Administrative Personnel 1 2012-01-01 2012-01-01 false What are the key activities of the COLA Advisory Committees? 591.241 Section 591.241 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL... Areas Program Administration § 591.241 What are the key activities of the COLA Advisory Committees?...

  5. 5 CFR 591.241 - What are the key activities of the COLA Advisory Committees?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 5 Administrative Personnel 1 2011-01-01 2011-01-01 false What are the key activities of the COLA Advisory Committees? 591.241 Section 591.241 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL... Areas Program Administration § 591.241 What are the key activities of the COLA Advisory Committees?...

  6. A QUICK KEY TO THE SUBFAMILIES AND GENERA OF ANTS OF THE SAVANNAH RIVER SITE, AIKEN, SC

    SciTech Connect

    Martin, D

    2006-10-04

    This taxonomic key was devised to support development of a Rapid Bioassessment Protocol using ants at the Savannah River Site. The emphasis is on ''rapid'' and, because the available keys contained a large number of genera not known to occur at the Savannah River Site, we found that the available keys were unwieldy. Because these keys contained more genera than we would likely encounter and because this larger number of genera required both more couplets in the key and often required examination of characters that are difficult to assess without higher magnifications (60X or higher) more time was required to process samples. In developing this set of keys I recognize that the character sets used may lead to some errors but I believe that the error rate will be small and, for the purpose of rapid bioassessment, this error rate will be acceptable provided that overall sample sizes are adequate. Oliver and Beattie (1996a, 1996b) found that for rapid assessment of biodiversity the same results were found when identifications were done to morphospecies by people with minimal expertise as when the same data sets were identified by subject matter experts. Basset et al. (2004) concluded that it was not as important to correctly identify all species as it was to be sure that the study included as many functional groups as possible. If your study requires high levels of accuracy, it is highly recommended that when you key out a specimen and have any doubts concerning the identification, you should refer to keys in Bolton (1994) or to the other keys used to develop this area specific taxonomic key.

  7. Identification of key residues involved in the activation and signaling properties of dopamine D3 receptor.

    PubMed

    Kota, Kokila; Kuzhikandathil, Eldo V; Afrasiabi, Milad; Lacy, Brett; Kontoyianni, Maria; Crider, A Michael; Song, Daniel

    2015-09-01

    The dopamine D3 receptor exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties that distinguish it from the closely-related D2 receptors. While amino acid residues important for D3 receptor ligand binding have been identified, the residues involved in activation of D3 receptor signaling and induction of signaling properties have not been determined. In this paper, we used cis and trans isomers of a novel D3 receptor agonist, 8-OH-PBZI, and site-directed mutagenesis to identify key residues involved in D3 receptor signaling function. Our results show that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the D3 receptor tolerance and SRT properties. We show that while both agonists require a subset of residues in the orthosteric binding site of D3 receptors for activation of the receptor, the ability of the two isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3 receptor. We also show that unlike cis-8-OH-PBZI, which is a partial agonist at the dopamine D2S receptor and full agonist at dopamine D2L receptor, trans-8-OH-PBZI is a full agonist at both D2S and D2L receptors. The different effect of the two isomers on D3 receptor signaling properties and D2S receptor activation correlated with differential effects of the isomers on agonist-induced mouse locomotor activity. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3 and D2 receptor homology modeling and for determining the role of D3 receptor tolerance and SRT properties in signaling and behavior. PMID:26116441

  8. The DOS 1 neutron dosimetry experiment at the HB-4-A key 7 surveillance site on the HFIR pressure vessel

    SciTech Connect

    Farrell, K.; Kam, F.B.; Baldwin, C.A.

    1994-01-01

    A comprehensive neutron dosimetry experiment was made at one of the prime surveillance sites at the High Flux Isotope Reactor (HFIR) pressure vessel to aid radiation embrittlement studies of the vessel and to benchmark neutron transport calculations. The thermal neutron flux at the key 7, position 5 site was found, from measurements of radioactivation of four cobalt wires and four silver wires, to be 2.4 {times} 10{sup 12} n{center_dot}m{sup {minus}2}{center_dot}s{sup {minus}1}. The thermal flux derived from two helium accumulation monitors was 2.3 {times} 10{sup 12} n{center_dot}m{sup {minus}2}{center_dot}{sup {minus}1}. The thermal flux estimated by neutron transport calculations was 3.7 {times} 10{sup 12} n{center_dot}m{sup {minus}2}s{sup {minus}1}. The fast flux, >1 MeV, determined from two nickel activation wires, was 1.5 {times} 10{sup 12} n{center_dot}m{sup {minus}2}{center_dot}s{sup {minus}1}, in keeping with values obtained earlier from stainless steel surveillance monitors and with a computed value of 1.2 {times} 10{sup 13} n{center_dot}m{sup {minus}2}{center_dot}{sup {minus}1}. The fast fluxes given by two reaction-product-type monitors, neptunium-237 and beryllium, were 2.6 {times} 10{sup 13} n{center_dot}m{sup {minus}2}{center_dot}s {sup {minus}1} and 2.2 {times} 10{sup 13} n{center_dot}m{sup {minus}2}s{sup {minus}1}, respectively. Follow-up experiments indicate that these latter high values of fast flux are reproducible but are false; they are due to the creation of greater levels of reaction products by photonuclear events induced by an exceptionally high ratio of gamma flux to fast neutron flux at the vessel.

  9. Computational Method for the Systematic Identification of Analog Series and Key Compounds Representing Series and Their Biological Activity Profiles.

    PubMed

    Stumpfe, Dagmar; Dimova, Dilyana; Bajorath, Jürgen

    2016-08-25

    A computational methodology is introduced for detecting all unique series of analogs in large compound data sets, regardless of chemical relationships between analogs. No prior knowledge of core structures or R-groups is required, which are automatically determined. The approach is based upon the generation of retrosynthetic matched molecular pairs and analog networks from which distinct series are isolated. The methodology was applied to systematically extract more than 17 000 distinct series from the ChEMBL database. For comparison, analog series were also isolated from screening compounds and drugs. Known biological activities were mapped to series from ChEMBL, and in more than 13 000 of these series, key compounds were identified that represented substitution sites of all analogs within a series and its complete activity profile. The analog series, key compounds, and activity profiles are made freely available as a resource for medicinal chemistry applications. PMID:27501131

  10. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth. PMID:26360629

  11. National Low-Level Waste Management Program final summary report of key activities and accomplishments for fiscal year 1997

    SciTech Connect

    Rittenberg, R.B.

    1998-03-01

    The US Department of Energy (DOE) has responsibilities under the Low-Level Radioactive Waste Policy Amendments Act of 1985 to assist states and compacts in their siting and licensing efforts for low-level radioactive waste disposal facilities. The National Low-Level Waste Management Program (NLLWMP) is the element of the DOE that performs the key support activities under the Act. The NLLWMP`s activities are driven by the needs of the states and compacts as they prepare to manage their low-level waste under the Act. Other work is added during the fiscal year as necessary to accommodate new requests brought on by status changes in states` and compacts` siting and licensing efforts. This report summarizes the activities and accomplishments of the NLLWMP during FY 1997.

  12. Identifying Key Features of Effective Active Learning: The Effects of Writing and Peer Discussion

    ERIC Educational Resources Information Center

    Linton, Debra L.; Pangle, Wiline M.; Wyatt, Kevin H.; Powell, Karli N.; Sherwood, Rachel E.

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between…

  13. Key to GHG fluxes from organic soils: site characteristics, agricultural practices or water table management?

    NASA Astrophysics Data System (ADS)

    Tiemeyer, Bärbel

    2015-04-01

    Drained peatlands are hotspots of greenhouse gas (GHG) emissions. Agriculture is the major land use type for peatlands in Germany and other European countries, but strongly varies in its intensity regarding the groundwater level and the agricultural management. Although the mean annual water table depth is sometimes proposed as an overall predictor for GHG emissions, there is a strong variability of its effects on different peatlands. Furthermore, re-wetting measures generally decrease carbon dioxide emissions, but may strongly increase methane emissions. We synthesized 250 annual GHG budgets for 120 different sites in 13 German peatlands. Carbon dioxide (net ecosystem exchange and ecosystem respiration), nitrous oxide and methane fluxes were measured with transparent and opaque manual chambers. Land management ranged from very intensive use with arable land or grassland with up to five cuts per year to partially or completely re-wetted peatlands. Besides the GHG fluxes, biomass yield, fertilisation, groundwater level, climatic data, vegetation composition and soil properties were measured. Overall, we found a large variability of the total GHG budget ranging from small uptakes to extremely high emissions (> 70 t CO2-equivalents/(ha yr)). At nearly all sites, carbon dioxide was the major component of the GHG budget. Site conditions, especially the nitrogen content of the unsaturated zone and the intra-annual water level distribution, controlled the GHG emissions of the agricultural sites. Although these factors are influenced by natural conditions (peat type, regional hydrology), they could be modified by an improved water management. Agricultural management such as the number of cuts had only a minor influence on the GHG budgets. At the level of individual peatlands, higher water levels always decreased carbon dioxide emissions. In nearly all cases, the trade-off between reduced carbon dioxide and increased methane emissions turned out in favour of the re

  14. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  15. Biological activity in Technosols as a key factor of their structure

    NASA Astrophysics Data System (ADS)

    Watteau, Françoise; Villemin, Geneviève; Bouchard, Adeline; Monserié, Marie-France; Séré, Geoffroy; Schwartz, Christophe; Morel, Jean-Louis

    2010-05-01

    The studies of the dynamics of organic matters within soils, show that their structural stability depends on the biological activity bound to the degradation of organic products. We wondered what it was for Technosols there. We then tried to specify the contribution of this biological activity to the structure of three contrasted technosols : - Technosol 1: a material originated from a former steel industry containing steel and coke residues, which was deposited two years ago in lysimetric plots - Technosol 2: a constructed soil (30 months) resulting from the combination of paper-mill sludge, thermally treated soil material excavated from a former coking plant site, and green-waste compost - Technosol 3: 30 years old technosol developed on flotation ponds of a former steel mill with strong metallic pollution, on which grows a forest ecosystem If these 3 technosols presented initially a similar organic carbon content (around 70 g.kg-1), the origin of organic matters was different A follow-up of the structural stability of these 3 systems, based on techniques of granulometric soil fractionation and morphological/analytical characterization at ultrastructural scale (TEM/EDX), was realized. Results showed the specific contribution of organic matters to the formation of stable organo-mineral associations, in particular those belonging to (0-50 μm) fraction. They mainly involved organic matter from vegetal origin coming from the spontaneous colonization of these 3 sites, but also from microbial origin corresponding to rhizospheric bacteria producing exopolymers. Organic matters from the compost and cellulosic fibers from the paper-mill sludge also contributed to the formation of organo-mineral associations all the more that compost was also a source of microorganisms. Organic matters were also associated to pollutant metallic elements (Pb, Zn, Mn) initially brought by the materials, then highlighting their possible transfer and questioning about their (bio

  16. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  17. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. PMID:26672454

  18. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  19. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  20. Amsterdamøya: a key site for the post-glacial of Svalbard

    NASA Astrophysics Data System (ADS)

    Bakke, Jostein; Balascio, Nicholas; van der Bilt, Willem; D`Andrea, William; Bradley, Raymond; Gjerde, Marthe; Hormes, Anne; Olafsdottir, Sædis; Røthe, Torgeir; Vasskog, Kristian; De Wet, Greg; Werner, Johannes

    2016-04-01

    No other place on Earth is changing as fast as the Arctic in terms of climate. On average this region is warming twice as fast as the global average with a seasonal bias towards winter. A major retreat in sea ice extent accompanied by an even more massive thinning represents one of the most robust trends in the Arctic. This trend is anticipated to continue in the decades to come and, according to some models, will leave the Arctic Ocean open during summer some time between 2050-2100. Unabated reduction in the spring-snow cover represents another significant trend. The current warming is also expressed in the massive melting of the Greenland ice sheet as well as local glaciers and ice caps in the Arctic, which causes increased freshwater influx to the Arctic Ocean and adjacent seas. Climate modeling and scenarios are improving and becoming of growing importance, but without a firmer understanding of natural climate variability over longer timescale it is still hard to evaluate and best read the output from these models. In the SHIFTS project we have done an unparalleled effort to overcome this quandary, providing necessary empirical data on past climate which is critical for assessing past changes in atmospheric circulation patterns controlling Arctic hydroclimate. Our study site is located at the northwestern corner of Svalbard on the Island of Amsterdamøya, a site sensitive to changes in both oceanic and atmospheric forcing, at tail of the westward moving branch of the North Atlantic current. Here we have cored several lakes with the goal of providing quantitative data on temperature, hydrology and winter precipitation for the Holocene. Our approach has been to combine reconstruction of glaciers with lipid biomarkers and hydrogen isotopes with the goal of unravel the underlying signature of past climate in the Arctic. Chronological control is secured by radiocarbon dates on macrofossils combined with measurement of paleomagnetic secular variations. Here we

  1. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  2. Prefrontal Cortical Network Connections: Key Site of Vulnerability in Stress and Schizophrenia

    PubMed Central

    Arnsten, Amy F.T.

    2011-01-01

    The symptoms of schizophrenia involve profound dysfunction of the prefrontal cortex (PFC). PFC networks create our “mental sketch pad”, and PFC dysfunction contributes to symptoms such as cognitive deficits, thought disorder, delusions and hallucinations. Neuropathological studies of schizophrenia have shown marked loss of dendritic spines in deep layer III, the sublayer where PFC microcircuits reside. The microcircuits consist of recurrent excitatory pyramidal cell networks that interconnect on spines, and excite each other via NMDA receptor signaling. The pyramidal cell persistent firing is sculpted by lateral inhibition from GABAergic basket and chandelier cells, thus creating tuned, persistent firing needed for accurate representational knowledge (i.e. working memory). The strength of pyramidal cell network connections is markedly and flexibly altered by intracellular signaling pathways in dendritic spines, a process called Dynamic Network Connectivity (DNC). DNC proteins such as HCN channels are concentrated on dendritic spines in deep layer III. Under optimal conditions, network inputs to pyramidal cells are strengthened by noradrenergic alpha-2A inhibition of cAMP-HCN channel signaling, and sculpted by dopamine D1-cAMP-HCN channel weakening of inappropriate inputs. However, with stress exposure, high levels of cAMP-HCN channel signaling produces a collapse in network firing. With chronic stress exposure, spines reduce in size and are lost, and this process involves increased PKC signaling. Importantly, molecules that normally strengthen PFC networks connections and/or reverse the stress response, are often genetically altered in schizophrenia. As exposure to stress is a key factor in the precipitation of schizophrenic symptoms, these dysregulated signaling pathways in deep layer III may interact with already vulnerable circuitry to cause spine loss and the descent into illness. PMID:21345366

  3. Comprehensive paleoseismic geological studies in a key site in southwestern Kola Peninsula (Northeast of the Fennoscandian Shield)

    NASA Astrophysics Data System (ADS)

    Nikolaeva, S. B.; Nikonov, A. A.; Shvarev, S. V.; Rodkin, M. V.

    2016-07-01

    This paper considers the results of detailed paleoseismic and geological investigations in a key site in the wall of the Imandra Lake depression (Kola Peninsula Region, Northeast of the Fennoscandian Shield). Study of different groups of paleoseismic deformations developed in the fault zone and the application of new methods and techniques made it possible to identify a large seismotectonic zone characterized by great earthquakes at the end of the Late Glacial and in the Holocene. The investigation data are indicative of the necessity to estimate the seismic potential in the Kola Atomic plant area in a different way.

  4. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  5. Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb, a Key Enzyme in Amylopectin Biosynthesis

    PubMed Central

    Makhmoudova, Amina; Williams, Declan; Brewer, Dyanne; Massey, Sarah; Patterson, Jenelle; Silva, Anjali; Vassall, Kenrick A.; Liu, Fushan; Subedi, Sanjeena; Harauz, George; Siu, K. W. Michael; Tetlow, Ian J.; Emes, Michael J.

    2014-01-01

    Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser649, Ser286, and Ser297. Two Ca2+-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser649 and Ser286 phosphorylation, and K2, responsible for Ser649 and Ser297 phosphorylation. The Ser286 and Ser297 phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser297 forms a stable salt bridge with Arg665, part of a conserved Cys-containing domain in plant branching enzymes. Ser649 conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application. PMID:24550386

  6. Key operational characteristics in emergency department observation units: a comparative study between sites in the United States and Asia

    PubMed Central

    2014-01-01

    Background To improve efficiency, emergency departments (EDs) use dedicated observation units (OUs) to manage patients who are unable to be discharged home, yet do not clearly require inpatient hospitalization. However, operational metrics and their ideal targets have not been created for this setting and patient population. Variation in these metrics across different countries has not previously been reported. This study aims to define and compare key operational characteristics between three ED OUs in the United States (US) and three ED OUs in Asia. Methods This is a descriptive study of six tertiary-care hospitals, all of which are level 1 trauma centers and have OUs managed by ED staff. We collected data via various methods, including a standardized survey, direct observation, and interviews with unit leadership, and compared these data across continents. Results We define multiple key operational characteristics to compare between sites, including OU length of stay (LOS), OU discharge rate, and bed turnover rate. OU LOS in the US and Asian sites averaged 12.9 hours (95% CI, 8.3 to 17.5) and 20.5 hours (95% CI, -49.4 to 90.4), respectively (P = 0.39). OU discharge rates in the US and Asia averaged 84.3% (95% CI, 81.5 to 87.2) and 88.7% (95% CI, 81.5 to 95.8), respectively (P = 0.11), and the bed turnover rates in the US and Asian sites averaged 1.6 patients/bed/day (95% CI, -0.1 to 3.3) and 0.9 patient/bed/day (95% CI, -0.6 to 2.4), respectively (P = 0.27). Conclusions Prior research has shown that the OU is a resource that can mitigate many of problems in the ED and hospital, while simultaneously improving patient care and satisfaction. We describe key operational characteristics that are relevant to all OUs, regardless of geography or healthcare system to monitor and maximize efficiency. Although measures of LOS and bed turnover varied widely between US and Asian sites, we did not find a statistically significant difference. Use of these metrics

  7. Key Sites for P2X Receptor Function and Multimerization: Overview of Mutagenesis Studies on a Structural Basis

    PubMed Central

    Hausmann, Ralf; Kless, Achim; Schmalzing, Günther

    2015-01-01

    P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-Å crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures. PMID:25439586

  8. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  9. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  10. Key programmatic steps and activities for implementing the Uranium Mill Tailings Remedial Action Project. [UMTRA Project

    SciTech Connect

    Not Available

    1985-07-01

    The Uranium Mill Tailings Radiation Control Act of 1978 (UMTRCA) was enacted based upon findings by Congress that uranium mill tailings located at active and inactive hazard to the public, and that protection of the public health, safety and welfare, and the regulations of interstate commerce, require that every reasonable effort be made to provide for the stabilization, disposal, and control in a safe and environmentally sound manner of such tailings in order to prevent or minimize radon diffusion into the environment and to prevent or minimize other environmental hazards from such tailings.'' A general understanding of the steps leading to elimination of the hazards associated with designated uranium mill tailings sites, and the parties involved in that effort, are presented in this document. A representative schedule is also presented in this document to show both program sequence and activity interdependence. Those activities that have the most potential to influence program duration, because of the significant amount of additional time that may be required, include identification and selection of a suitable site, field data collection delays due to weather, actual acquisition of the designated or alternate disposal site, construction delays due to weather, and site licensing. This document provides an understanding of the steps, the sequence, the parties involved, and a representative duration of activities leading to remedial action and cleanup at the designated inactive uranium mill tailings sites. 10 refs., 5 figs., 1 tab.

  11. Experimental demonstration of an active phase randomization and monitor module for quantum key distribution

    NASA Astrophysics Data System (ADS)

    Sun, Shi-Hai; Liang, Lin-Mei

    2012-08-01

    Phase randomization is a very important assumption in the BB84 quantum key distribution (QKD) system with weak coherent source; otherwise, eavesdropper may spy the final key. In this Letter, a stable and monitored active phase randomization scheme for the one-way and two-way QKD system is proposed and demonstrated in experiments. Furthermore, our scheme gives an easy way for Alice to monitor the degree of randomization in experiments. Therefore, we expect our scheme to become a standard part in future QKD systems due to its secure significance and feasibility.

  12. The active site behaviour of electrochemically synthesised gold nanomaterials.

    PubMed

    Plowman, Blake J; O'Mullane, Anthony P; Bhargava, Suresh K

    2011-01-01

    Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces. PMID:22455038

  13. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  14. Prediction of Geomagnetic Activity and Key Parameters in High-Latitude Ionosphere-Basic Elements

    NASA Technical Reports Server (NTRS)

    Lyatsky, W.; Khazanov, G. V.

    2007-01-01

    Prediction of geomagnetic activity and related events in the Earth's magnetosphere and ionosphere is an important task of the Space Weather program. Prediction reliability is dependent on the prediction method and elements included in the prediction scheme. Two main elements are a suitable geomagnetic activity index and coupling function -- the combination of solar wind parameters providing the best correlation between upstream solar wind data and geomagnetic activity. The appropriate choice of these two elements is imperative for any reliable prediction model. The purpose of this work was to elaborate on these two elements -- the appropriate geomagnetic activity index and the coupling function -- and investigate the opportunity to improve the reliability of the prediction of geomagnetic activity and other events in the Earth's magnetosphere. The new polar magnetic index of geomagnetic activity and the new version of the coupling function lead to a significant increase in the reliability of predicting the geomagnetic activity and some key parameters, such as cross-polar cap voltage and total Joule heating in high-latitude ionosphere, which play a very important role in the development of geomagnetic and other activity in the Earth s magnetosphere, and are widely used as key input parameters in modeling magnetospheric, ionospheric, and thermospheric processes.

  15. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

  16. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  17. Chew Bahir: A Key Site within the Hominin Sites and Paleolakes Drilling Project, towards a Half Million-Year Climate Record from Southern Ethiopia

    NASA Astrophysics Data System (ADS)

    Schaebitz, F.; Asrat, A.; Lamb, H. F.; Trauth, M. H.; Foerster, V. E.; Junginger, A.; Raub, T. D.; Gromig, R.; Viehberg, F. A.; Roberts, H. M.; Cohen, A.

    2015-12-01

    Chew Bahir, a saline mudflat today, is one of the five sites in East Africa, drilled within the framework of HSPDP (Hominin Site and Paleolakes Drilling Project). It is also one of the key sites of the Collaborative Research Centre (CRC-806) "Our way to Europe" aiming at the reconstruction of environmental conditions in the source region of modern man (H. sapiens). It is suggested that a changing environment could have triggered the mobility and dispersal of modern man. The oldest known fossils of anatomical modern humans (~195 ka BP) were found in the Omo basin, not more than 90km westwards of our drill site. The deposits in the tectonic basin of Chew Bahir in southern Ethiopia were cored in Nov. 2014 in two boreholes down to 280 m and 260 m below surface respectively. The overlapping long cores (drilled ~20 m apart from each other), were opened, scanned, described and sampled in low resolution in April 2015. The recovered sediments mostly contain green-greyish to light coloured and brown to reddish clays and silty clays, interbedded with some laminated mica-rich sand layers and occurrences of carbonate concretions and nodules, which decrease upcore. Here we will present a first set of results on the composite core, comprising mainly lithology and magnetic susceptibility (MS). Based on known sedimentation rates from pre-studies performed on short cores across the basin, we anticipate the deep drilled cores to cover at least 500 ka BP. Moreover, new insights into the role of post-depositional alteration, especially of clay minerals and zeolites, will be presented as a contribution to an improved understanding of formation processes. The results support the identification of wet and dry climate periods in the past. Those pronounced variations of moisture availability, are thought to have influenced the evolution and mobility of Homo sapiens sapiens.

  18. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  19. Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

    PubMed

    Zhuang, X; Wang, Q; Wang, B; Zhong, T; Cao, Y; Li, K; Yin, J

    2014-06-01

    hydrogen bonding that acts as a holder. So we believe that Tyr111 of HoblOBP1 is a key binding site. We also believe that Ile80A is a very important binding site, especially to some ligands. PMID:24576058

  20. Site Selection for Mars Surveyor Landing Sites: Some Key Factors for 2001 and Relation to Long-Term Exploration of Mars

    NASA Astrophysics Data System (ADS)

    Head, James W.

    1999-01-01

    objectives can in principle be achieved through the exploration of a site meeting the basic engineering constraints. Armed with all of this important background information, one can then proceed to (4) the selection of optimum sites to address major scientific and programmatic objectives. Following the successful completion of this process and the selection of a site or region, there is a further step of mission optimization, in which a detailed mission profile and surface exploration plan is developed. In practice, the process never works in a linear fashion. Scientific goals are influenced by ongoing discoveries and developments and simple crystallization of thinking. Programmatic goals are influenced by evolving fiscal constraints, perspectives on program duration, and roles of specific missions in the context of the larger program. Engineering constraints are influenced by evolving fiscal constraints, decisions on hardware design that may have little to do with scientific goals (e.g., lander clearance; size of landing ellipse), and evolving understanding (e.g., assessment of engineering constraint space reveals further the degree to which mission duration is severely influenced by available solar energy and thus latitude). Lander scientific payload is influenced by fiscal constraints, total mass, evolving complexity, technological developments, and a payload selection process that may involve very long-term goals (e.g., human exploration) as well as shorter term scientific and programmatic goals. Site selection activities commonly involve scientists who are actively trying to decipher the complex geology of the crust of Mars and to unravel its geologic history through geological mapping. By the nature of the process, they are thinking in terms of broad morphostratigraphic units which may have multiple possible origins, defined using images with resolutions of many tens to hundreds of meters, and whose surfaces at the scale of the lander and rover are virtually unknown

  1. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  2. Investigation of the relationship between atmospheric mercury and concentrations of key greenhouse gases at a mountainous monitoring site.

    PubMed

    Kim, Ki-Hyun; Pandey, Sudhir Kumar; Brown, Richard J C; Sheu, Guey Rong; Jeon, Eui-Chan; Jung, Kweon; Kang, Chang-Hee

    2015-03-01

    The concentration of total gaseous mercury (TGM) was monitored, together with some key greenhouse gases (GHGs: carbon dioxide (CO2), methane (CH4), and water (H2O) vapor) at hourly intervals at a mountainous monitoring site close to the highly industrialized city of Seoul, Korea. Correlations between the concentrations of Hg and those of the greenhouse gases were examined to assess their source characteristics and responses to changes in meteorological conditions. The mean Hg levels in this study (3.58 ± 2.13 ng m(-3)) were considerably lower (by, e.g., 24.3%) than those measured previously in other comparable sites during 1999-2006 (4.73 ± 1.34 ng m(-3)). Accordingly, such a reduction in Hg levels suggests the effectiveness of the regulatory measures enforced over the years. The mean Hg level observed in this study is also lower (by approximately 5%) than those in other Asian locations. In contrast, the mean concentrations of the two most important GHGs (CO2 and CH4) were moderately higher than those of other locations across the world (by approximately 4-9%). The results of our analysis indicate that the behavior of Hg is strongly correlated with water vapor and CH4 in terms of their source characteristics, despite notable differences in their diurnal patterns. PMID:25639653

  3. 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

  4. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2domains reveal that the (HhH)2domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  5. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  6. Quantification of key long-term risks at CO₂ sequestration sites: Latest results from US DOE's National Risk Assessment Partnership (NRAP) Project

    SciTech Connect

    Pawar, Rajesh; Bromhal, Grant; Carroll, Susan; Chu, Shaoping; Dilmore, Robert; Gastelum, Jason; Oldenburg, Curt; Stauffer, Philip; Zhang, Yingqi; Guthrie, George

    2014-12-31

    Risk assessment for geologic CO₂ storage including quantification of risks is an area of active investigation. The National Risk Assessment Partnership (NRAP) is a US-Department of Energy (US-DOE) effort focused on developing a defensible, science-based methodology and platform for quantifying risk profiles at geologic CO₂ sequestration sites. NRAP has been developing a methodology that centers round development of an integrated assessment model (IAM) using system modeling approach to quantify risks and risk profiles. The IAM has been used to calculate risk profiles with a few key potential impacts due to potential CO₂ and brine leakage. The simulation results are also used to determine long-term storage security relationships and compare the long-term storage effectiveness to IPCC storage permanence goal. Additionally, we also demonstrate application of IAM for uncertainty quantification in order to determine parameters to which the uncertainty in model results is most sensitive.

  7. Quantification of key long-term risks at CO₂ sequestration sites: Latest results from US DOE's National Risk Assessment Partnership (NRAP) Project

    DOE PAGESBeta

    Pawar, Rajesh; Bromhal, Grant; Carroll, Susan; Chu, Shaoping; Dilmore, Robert; Gastelum, Jason; Oldenburg, Curt; Stauffer, Philip; Zhang, Yingqi; Guthrie, George

    2014-12-31

    Risk assessment for geologic CO₂ storage including quantification of risks is an area of active investigation. The National Risk Assessment Partnership (NRAP) is a US-Department of Energy (US-DOE) effort focused on developing a defensible, science-based methodology and platform for quantifying risk profiles at geologic CO₂ sequestration sites. NRAP has been developing a methodology that centers round development of an integrated assessment model (IAM) using system modeling approach to quantify risks and risk profiles. The IAM has been used to calculate risk profiles with a few key potential impacts due to potential CO₂ and brine leakage. The simulation results are alsomore » used to determine long-term storage security relationships and compare the long-term storage effectiveness to IPCC storage permanence goal. Additionally, we also demonstrate application of IAM for uncertainty quantification in order to determine parameters to which the uncertainty in model results is most sensitive.« less

  8. Investigation of key quantities for the first indirect aerosol effect contrasting MSG SEVIRI and ground site measurements

    NASA Astrophysics Data System (ADS)

    Merk, Daniel; Deneke, Hartwig; Pospichal, Bernhard; Seifert, Patric; Ansmann, Albert

    2014-05-01

    The first indirect aerosol effect remains one of the main uncertainties in projections of anthropogenic climate change. Satellites provide a unique possibility to globally quantify the importance of the first indirect aerosol effect. Given a constant liquid water content within the cloud, a higher cloud droplet number concentration results in higher cloud albedo. But the cloud albedo is also altered by the geometrical cloud extent. Therefore the two key quantities for this investigation are the CDNC and the geometrical cloud extent. Both quantities can not be obtained directly from current geostationary satellites. Due to necessary assumptions and missing information about the vertical cloud structure, the retrieval of both quantities remains a great challenge. Our aim is to investigate the accuracy of current satellite retrievals by contrasting the key quantities with those obtained from ground-site. The satellite retrieval is based on the method described by Nakajima and King to derive the optical cloud depth and the effective radius. In a second step, the CDNC and cloud extent is determined, assuming an adiabatically increasing liquid water content above cloud base. Single-layer liquid water clouds are simultaneously observed with ground-based remote sensing instruments at different locations in Germany (Leipzig, Juelich, Melpitz). We use a ceilometer to detect the cloud base height, a 35 GHz cloud radar to detect the cloud top height and the reflectivity profile, and a microwave radiometer to obtain the liquid water path. We developed an Optimal Estimation approach to retrieve the CDNC as well as the liquid water content profile. Our observation vector consists of the radar reflectivity profile and the liquid water path. We compare the retrieved quantities from ground with the satellite perspective to closer investigate the assumption of adiabatic cloud profiles.

  9. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  10. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  11. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

    Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed. PMID:4942834

  12. The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes.

    PubMed

    Beumer, Wouter; Gibney, Sinead M; Drexhage, Roosmarijn C; Pont-Lezica, Lorena; Doorduin, Janine; Klein, Hans C; Steiner, Johann; Connor, Thomas J; Harkin, Andrew; Versnel, Marjan A; Drexhage, Hemmo A

    2012-11-01

    This review describes a key role for mononuclear phagocytes in the pathogenesis of major psychiatric disorders. There is accumulating evidence for activation of microglia (histopathology and PET scans) and circulating monocytes (enhanced gene expression of immune genes, an overproduction of monocyte/macrophage-related cytokines) in patients with bipolar disorder, major depressive disorder, and schizophrenia. These data are strengthened by observations in animal models, such as the MIA models, the chronic stress models, and the NOD mouse model. In these animal models of depressive-, anxiety-, and schizophrenia-like behavior, similar activations of microglia and circulating monocytes can be found. These animal models also make in-depth pathogenic studies possible and show that microglia activation impacts neuronal development and function in brain areas congruent with the altered depressive and schizophrenia-like behaviors. PMID:22875882

  13. Identifying Key Features of Effective Active Learning: The Effects of Writing and Peer Discussion

    PubMed Central

    Pangle, Wiline M.; Wyatt, Kevin H.; Powell, Karli N.; Sherwood, Rachel E.

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between three sections taught by three different instructors in a full factorial design. The data set was analyzed by generalized linear mixed-effect models with three independent variables: student aptitude, treatment, and instructor, and three dependent (assessment) variables: change in score on pre- and postactivity clicker questions, and coding scores on in-class writing and exam essays. All independent variables had significant effects on student performance for at least one of the dependent variables. Students with higher aptitude scored higher on all assessments. Student scores were higher on exam essay questions when the activity was implemented with a writing component compared with peer discussion only. There was a significant effect of instructor, with instructors showing different degrees of effectiveness with active-learning techniques. We suggest that individual writing should be implemented as part of active learning whenever possible and that instructors may need training and practice to become effective with active learning. PMID:25185230

  14. Identifying key features of effective active learning: the effects of writing and peer discussion.

    PubMed

    Linton, Debra L; Pangle, Wiline M; Wyatt, Kevin H; Powell, Karli N; Sherwood, Rachel E

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between three sections taught by three different instructors in a full factorial design. The data set was analyzed by generalized linear mixed-effect models with three independent variables: student aptitude, treatment, and instructor, and three dependent (assessment) variables: change in score on pre- and postactivity clicker questions, and coding scores on in-class writing and exam essays. All independent variables had significant effects on student performance for at least one of the dependent variables. Students with higher aptitude scored higher on all assessments. Student scores were higher on exam essay questions when the activity was implemented with a writing component compared with peer discussion only. There was a significant effect of instructor, with instructors showing different degrees of effectiveness with active-learning techniques. We suggest that individual writing should be implemented as part of active learning whenever possible and that instructors may need training and practice to become effective with active learning. PMID:25185230

  15. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  16. Directing reaction pathways by catalyst active-site selection using self-assembled monolayers.

    PubMed

    Pang, Simon H; Schoenbaum, Carolyn A; Schwartz, Daniel K; Medlin, J Will

    2013-01-01

    One key route for controlling reaction selectivity in heterogeneous catalysis is to prepare catalysts that exhibit only specific types of sites required for desired product formation. Here we show that alkanethiolate self-assembled monolayers with varying surface densities can be used to tune selectivity to desired hydrogenation and hydrodeoxygenation products during the reaction of furfural on supported palladium catalysts. Vibrational spectroscopic studies demonstrate that the selectivity improvement is achieved by controlling the availability of specific sites for the hydrogenation of furfural on supported palladium catalysts through the selection of an appropriate alkanethiolate. Increasing self-assembled monolayer density by controlling the steric bulk of the organic tail ligand restricts adsorption on terrace sites and dramatically increases selectivity to desired products furfuryl alcohol and methylfuran. This technique of active-site selection simultaneously serves both to enhance selectivity and provide insight into the reaction mechanism. PMID:24025780

  17. The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion

    NASA Astrophysics Data System (ADS)

    Giachin, Gabriele; Mai, Phuong Thao; Tran, Thanh Hoa; Salzano, Giulia; Benetti, Federico; Migliorati, Valentina; Arcovito, Alessandro; Longa, Stefano Della; Mancini, Giordano; D'Angelo, Paola; Legname, Giuseppe

    2015-10-01

    The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

  18. The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion

    PubMed Central

    Giachin, Gabriele; Mai, Phuong Thao; Tran, Thanh Hoa; Salzano, Giulia; Benetti, Federico; Migliorati, Valentina; Arcovito, Alessandro; Longa, Stefano Della; Mancini, Giordano; D’Angelo, Paola; Legname, Giuseppe

    2015-01-01

    The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation. PMID:26482532

  19. Key findings from HSC's 2010 site visits: health care markets weather economic downturn, brace for health reform.

    PubMed

    Felland, Laurie E; Grossman, Joy M; Tu, Ha T

    2011-05-01

    Lingering fallout--loss of jobs and employer coverage--from the great recession slowed demand for health care services but did little to slow aggressive competition by dominant hospital systems for well-insured patients, according to key findings from the Center for Studying Health System Change's (HSC) 2010 site visits to 12 nationally representative metropolitan communities. Hospitals with significant market clout continued to command high payment rate increases from private insurers, and tighter hospital-physician alignment heightened concerns about growing provider market power. High and rising premiums led to increasing employer adoption of consumer-driven health plans and continued increases in patient cost sharing, but the broader movement to educate and engage consumers in care decisions did not keep pace. State and local budget deficits led to some funding cuts for safety net providers, but an influx of federal stimulus funds increased support to community health centers and shored up Medicaid programs, allowing many people who lost private insurance because of job losses to remain covered. Hospitals, physicians and insurers generally viewed health reform coverage expansions favorably, but all worried about protecting revenues as reform requirements phase in. PMID:21614861

  20. Rat intestinal trehalase. Studies of the active site.

    PubMed

    Chen, C C; Guo, W J; Isselbacher, K J

    1987-11-01

    Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed. PMID:3426558

  1. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    PubMed Central

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

    2015-01-01

    5′-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. PMID:25806409

  2. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  3. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  4. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  5. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  6. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  7. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  8. Phosphatase activity in relation to key litter and soil properties in mature subtropical forests in China.

    PubMed

    Hou, Enqing; Chen, Chengrong; Wen, Dazhi; Liu, Xian

    2015-05-15

    Phosphatase-mediated phosphorus (P) mineralization is one of the critical processes in biogeochemical cycling of P and determines soil P availability in forest ecosystems; however, the regulation of soil phosphatase activity remains elusive. This study investigated the potential extracellular activities of acid phosphomonoesterase (AcPME) and phosphodiesterase (PDE) and how they were related to key edaphic properties in the L horizon (undecomposed litter) and F/H horizon (fermented and humified litter) and the underlying mineral soil at the 0-15cm depth in eight mature subtropical forests in China. AcPME activity decreased significantly in the order of F/H horizon>L horizon>mineral soil horizon, while the order for PDE activity was L horizon=F/H horizon>mineral soil horizon. AcPME (X axis) and PDE (Y axis) activities were positively correlated in all horizons with significantly higher slope in the L and F/H horizons than in the mineral soil horizon. Both AcPME and PDE activities were positively related to microbial biomass C, moisture content and water-holding capacity in the L horizon, and were positively related to soil C:P, N:P and C:N ratios and fine root (diameter≤2mm) biomass in the mineral soil horizon. Both enzyme activities were also interactively affected by forest and horizon, partly due to the interactive effect of forest and horizon on microbial biomass. Our results suggest that modulator(s) of the potential extracellular activity of phosphatases vary with horizon, depending on the relative C, P and water availability of the horizon. PMID:25700362

  9. Identifying key controls on the behavior of an acidic-U(VI) plume in the Savannah River Site using reactive transport modeling.

    PubMed

    Bea, Sergio A; Wainwright, Haruko; Spycher, Nicolas; Faybishenko, Boris; Hubbard, Susan S; Denham, Miles E

    2013-08-01

    Acidic low-level waste radioactive waste solutions were discharged to three unlined seepage basins at the F-Area of the Department of Energy (DOE) Savannah River Site (SRS), South Carolina, USA, from 1955 through 1989. Despite many years of active remediation, the groundwater remains acidic and contaminated with significant levels of U(VI) and other radionuclides. Monitored Natural Attenuation (MNA) is a desired closure strategy for the site, based on the premise that regional flow of clean background groundwater will eventually neutralize the groundwater acidity, immobilizing U(VI) through adsorption. An in situ treatment system is currently in place to accelerate this in the downgradient portion of the plume and similar measures could be taken upgradient if necessary. Understanding the long-term pH and U(VI) adsorption behavior at the site is critical to assess feasibility of MNA along with the in-situ remediation treatments. This paper presents a reactive transport (RT) model and uncertainty quantification (UQ) analyses to explore key controls on the U(VI)-plume evolution and long-term mobility at this site. Two-dimensional numerical RT simulations are run including the saturated and unsaturated (vadose) zones, U(VI) and H(+) adsorption (surface complexation) onto sediments, dissolution and precipitation of Al and Fe minerals, and key hydrodynamic processes are considered. UQ techniques are applied using a new open-source tool that is part of the developing ASCEM reactive transport modeling and analysis framework to: (1) identify the complex physical and geochemical processes that control the U(VI) plume migration in the pH range where the plume is highly mobile, (2) evaluate those physical and geochemical parameters that are most controlling, and (3) predict the future plume evolution constrained by historical, chemical and hydrological data. The RT simulation results show a good agreement with the observed historical pH and concentrations of U(VI), nitrates

  10. Identifying key controls on the behavior of an acidic-U(VI) plume in the Savannah River Site using reactive transport modeling

    NASA Astrophysics Data System (ADS)

    Bea, Sergio A.; Wainwright, Haruko; Spycher, Nicolas; Faybishenko, Boris; Hubbard, Susan S.; Denham, Miles E.

    2013-08-01

    Acidic low-level waste radioactive waste solutions were discharged to three unlined seepage basins at the F-Area of the Department of Energy (DOE) Savannah River Site (SRS), South Carolina, USA, from 1955 through 1989. Despite many years of active remediation, the groundwater remains acidic and contaminated with significant levels of U(VI) and other radionuclides. Monitored Natural Attenuation (MNA) is a desired closure strategy for the site, based on the premise that regional flow of clean background groundwater will eventually neutralize the groundwater acidity, immobilizing U(VI) through adsorption. An in situ treatment system is currently in place to accelerate this in the downgradient portion of the plume and similar measures could be taken upgradient if necessary. Understanding the long-term pH and U(VI) adsorption behavior at the site is critical to assess feasibility of MNA along with the in-situ remediation treatments. This paper presents a reactive transport (RT) model and uncertainty quantification (UQ) analyses to explore key controls on the U(VI)-plume evolution and long-term mobility at this site. Two-dimensional numerical RT simulations are run including the saturated and unsaturated (vadose) zones, U(VI) and H+ adsorption (surface complexation) onto sediments, dissolution and precipitation of Al and Fe minerals, and key hydrodynamic processes are considered. UQ techniques are applied using a new open-source tool that is part of the developing ASCEM reactive transport modeling and analysis framework to: (1) identify the complex physical and geochemical processes that control the U(VI) plume migration in the pH range where the plume is highly mobile, (2) evaluate those physical and geochemical parameters that are most controlling, and (3) predict the future plume evolution constrained by historical, chemical and hydrological data. The RT simulation results show a good agreement with the observed historical pH and concentrations of U(VI), nitrates and

  11. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  12. Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

    PubMed Central

    Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A.; Panté, Nelly; Kann, Michael

    2013-01-01

    Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis. PMID:24204256

  13. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    SciTech Connect

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A.; Cheng, Yong; Jain, Deepti; Visel, Axel; Pennacchio, Len A.; Weiss, Mitchell J.; Blobel, Gerd A.; Hardison, Ross C.

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

  14. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    DOE PAGESBeta

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A.; Cheng, Yong; Jain, Deepti; Visel, Axel; et al

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the resultsmore » of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.« less

  15. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  16. Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor

    PubMed Central

    Koizumi, Taichi; Terada, Tohru; Nakajima, Ken-ichiro; Kojima, Masaki; Koshiba, Seizo; Matsumura, Yoshitaka; Kaneda, Kohei; Asakura, Tomiko; Shimizu-Ibuka, Akiko; Abe, Keiko; Misaka, Takumi

    2015-01-01

    Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface. PMID:26263392

  17. Substrate Binding and Active Site Residues in RNases E and G

    PubMed Central

    Garrey, Stephen M.; Blech, Michaela; Riffell, Jenna L.; Hankins, Janet S.; Stickney, Leigh M.; Diver, Melinda; Hsu, Ying-Han Roger; Kunanithy, Vitharani; Mackie, George A.

    2009-01-01

    The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5′-sensor, the S1 domain, and the Mg+2/Mn+2 binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the kcat decrease ∼40- to 100-fold) and RNA binding consistent with a key role for the 5′-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition. PMID:19778900

  18. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

    Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation. PMID:9060667

  19. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  20. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  1. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  2. A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity

    PubMed Central

    Gwyn, Lori M.; Peak, Mandy M.; De, Pallabi; Rahman, Negar S.; Rodgers, Karla K.

    2009-01-01

    The recombination activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn2+ ions in its catalytically-required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well-resolved. To address this issue, we determined the stoichiometry of Zn2+ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn2+ ions. Stripping the full complement of bound Zn2+ ions to produce apo-protein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn2+ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn2+ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn2+ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active site residue E962 reduces Zn2+ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn2+ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962. PMID:19500590

  3. Src activation by β-adrenoreceptors is a key switch for tumor metastasis

    PubMed Central

    Armaiz-Pena, Guillermo N.; Allen, Julie K.; Cruz, Anthony; Stone, Rebecca L.; Nick, Alpa M.; Lin, Yvonne G.; Han, Liz Y.; Mangala, Lingegowda S.; Villares, Gabriel J.; Vivas-Mejia, Pablo; Rodriguez-Aquayo, Cristian; Nagaraja, Archana S.; Gharpure, Kshipra M.; Wu, Zheng; English, Robert D.; Soman, Kizhake V.; Shazhad, Mian M. K.; Zigler, Maya; Deavers, Michael T.; Zien, Alexander; Soldatos, Theodoros G.; Jackson, David B.; Wiktorowicz, John E.; Torres-Lugo, Madeline; Young, Tom; De Geest, Koen; Gallick, Gary E.; Bar-Eli, Menashe; Lopez-Berestein, Gabriel; Cole, Steve W.; Lopez, Gustavo E.; Lutgendorf, Susan K.; Sood, Anil K.

    2012-01-01

    Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment. PMID:23360994

  4. Src activation by β-adrenoreceptors is a key switch for tumour metastasis.

    PubMed

    Armaiz-Pena, Guillermo N; Allen, Julie K; Cruz, Anthony; Stone, Rebecca L; Nick, Alpa M; Lin, Yvonne G; Han, Liz Y; Mangala, Lingegowda S; Villares, Gabriel J; Vivas-Mejia, Pablo; Rodriguez-Aguayo, Cristian; Nagaraja, Archana S; Gharpure, Kshipra M; Wu, Zheng; English, Robert D; Soman, Kizhake V; Shahzad, Mian M K; Shazhad, Mian M K; Zigler, Maya; Deavers, Michael T; Zien, Alexander; Soldatos, Theodoros G; Jackson, David B; Wiktorowicz, John E; Torres-Lugo, Madeline; Young, Tom; De Geest, Koen; Gallick, Gary E; Bar-Eli, Menashe; Lopez-Berestein, Gabriel; Cole, Steve W; Lopez, Gustavo E; Lutgendorf, Susan K; Sood, Anil K

    2013-01-01

    Noradrenaline can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here we identify Src as a key regulator of phosphoproteomic signalling networks activated in response to beta-adrenergic signalling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumour cell migration, invasion and growth. In human ovarian cancer samples, high tumoural noradrenaline levels were correlated with high pSrc(Y419) levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signalling in the tumour microenvironment. PMID:23360994

  5. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  6. The linker connecting the two kringles plays a key role in prothrombin activation

    PubMed Central

    Pozzi, Nicola; Chen, Zhiwei; Pelc, Leslie A.; Shropshire, Daniel B.; Di Cera, Enrico

    2014-01-01

    The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146–167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146–167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146–167. PMID:24821807

  7. Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer

    PubMed Central

    Wei, Shuo; Kozono, Shingo; Kats, Lev; Nechama, Morris; Li, Wenzong; Guarnerio, Jlenia; Luo, Manli; You, Mi-Hyeon; Yao, Yandan; Kondo, Asami; Hu, Hai; Bozkurt, Gunes; Moerke, Nathan J.; Cao, Shugeng; Reschke, Markus; Chen, Chun-Hau; Rego, Eduardo M.; LoCoco, Francesco; Cantley, Lewis; Lee, Tae Ho; Wu, Hao; Zhang, Yan; Pandolfi, Pier Paolo; Zhou, Xiao Zhen; Lu, Kun Ping

    2015-01-01

    A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency. Using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but its drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the fusion oncogene PML-RARα and treats APL in cell and animal models and human patients. ATRA-induced Pin1 ablation also inhibits triple negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors. PMID:25849135

  8. Sustained impact of community-based physical activity interventions: key elements for success

    PubMed Central

    2013-01-01

    Background Compelling evidence supports the cost effectiveness and potential impact of physical activity on chronic disease prevention and health promotion. Quality of evidence is one piece, but certainly not the sole determinant of whether public health interventions, physical activity focused or otherwise, achieve their full potential for impact. Health promotion at both population and community levels must progress beyond health intervention models that isolate individuals from social, environmental, and political systems of influence. We offer a critical evaluation of lessons learned from two successful research initiatives to provide insights as to how health promotion research contributes to sustained impact. We highlight factors key to success including the theoretical and methodological integration of: i) a social ecological approach; ii) participatory action research (PAR) methods; and iii) an interdisciplinary team. Methods To identify and illustrate the key elements of our success we layered an evaluation of steps taken atop a review of relevant literature. Results In the school-based case study (Action Schools! BC), the success of our approach included early and sustained engagement with a broad cross-section of stakeholders, establishing partnerships across sectors and at different levels of government, and team members across multiple disciplines. In the neighbourhood built environment case study, the three domains guided our approach through study design and team development, and the integration of older adults’ perspectives into greenway design plans. In each case study we describe how elements of the domains serve as a guide for our work. Conclusion To sustain and maximize the impact of community-based public health interventions we propose the integration of elements from three domains of research that acknowledge the interplay between social, environmental and poilitical systems of influence. We emphasize that a number of key factors determine

  9. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  10. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  11. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  12. Transcriptome Analysis of Tomato Flower Pedicel Tissues Reveals Abscission Zone-Specific Modulation of Key Meristem Activity Genes

    PubMed Central

    Sun, Xiuli; Zhang, Rongzhi; Wu, Liang; Liang, Yanchun; Mao, Long

    2013-01-01

    Tomato flower abscises at the anatomically distinct abscission zone that separates the pedicel into basal and apical portions. During abscission, cell separation occurs only at the abscission zone indicating distinctive molecular regulation in its cells. We conducted a transcriptome analysis of tomato pedicel tissues during ethylene promoted abscission. We found that the abscission zone was the most active site with the largest set of differentially expressed genes when compared with basal and apical portions. Gene Ontology analyses revealed enriched transcription regulation and hydrolase activities in the abscission zone. We also demonstrate coordinated responses of hormone and cell wall related genes. Besides, a number of ESTs representing homologs of key Arabidopsis shoot apical meristem activity genes were found to be preferentially expressed in the abscission zone, including WUSCHEL (WUS), KNAT6, LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1(LBD1), and BELL-like homeodomain protein 1 (BLH1), as well as tomato axillary meristem genes BLIND (Bl) and LATERAL SUPPRESSOR (Ls). More interestingly, the homologs of WUS and the potential functional partner OVATE FAMILIY PROTEIN (OFP) were subsequently down regulated during abscission while Bl and AGL12 were continuously and specifically induced in the abscission zone. The expression patterns of meristem activity genes corroborate the idea that cells of the abscission zone confer meristem-like nature and coincide with the course of abscission and post-abscission cell differentiation. Our data therefore propose a possible regulatory scheme in tomato involving meristem genes that may be required not only for the abscission zone development, but also for abscission. PMID:23390523

  13. Transcriptome analysis of tomato flower pedicel tissues reveals abscission zone-specific modulation of key meristem activity genes.

    PubMed

    Wang, Xiang; Liu, Danmei; Li, Aili; Sun, Xiuli; Zhang, Rongzhi; Wu, Liang; Liang, Yanchun; Mao, Long

    2013-01-01

    Tomato flower abscises at the anatomically distinct abscission zone that separates the pedicel into basal and apical portions. During abscission, cell separation occurs only at the abscission zone indicating distinctive molecular regulation in its cells. We conducted a transcriptome analysis of tomato pedicel tissues during ethylene promoted abscission. We found that the abscission zone was the most active site with the largest set of differentially expressed genes when compared with basal and apical portions. Gene Ontology analyses revealed enriched transcription regulation and hydrolase activities in the abscission zone. We also demonstrate coordinated responses of hormone and cell wall related genes. Besides, a number of ESTs representing homologs of key Arabidopsis shoot apical meristem activity genes were found to be preferentially expressed in the abscission zone, including WUSCHEL (WUS), KNAT6, LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1(LBD1), and BELL-like homeodomain protein 1 (BLH1), as well as tomato axillary meristem genes BLIND (Bl) and LATERAL SUPPRESSOR (Ls). More interestingly, the homologs of WUS and the potential functional partner OVATE FAMILIY PROTEIN (OFP) were subsequently down regulated during abscission while Bl and AGL12 were continuously and specifically induced in the abscission zone. The expression patterns of meristem activity genes corroborate the idea that cells of the abscission zone confer meristem-like nature and coincide with the course of abscission and post-abscission cell differentiation. Our data therefore propose a possible regulatory scheme in tomato involving meristem genes that may be required not only for the abscission zone development, but also for abscission. PMID:23390523

  14. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  15. Autocrine abscisic acid plays a key role in quartz-induced macrophage activation.

    PubMed

    Magnone, Mirko; Sturla, Laura; Jacchetti, Emanuela; Scarfì, Sonia; Bruzzone, Santina; Usai, Cesare; Guida, Lucrezia; Salis, Annalisa; Damonte, Gianluca; De Flora, Antonio; Zocchi, Elena

    2012-03-01

    Inhalation of quartz induces silicosis, a lung disease where alveolar macrophages release inflammatory mediators, including prostaglandin-E(2) (PGE(2)) and tumor necrosis factor α (TNF-α). Here we report the pivotal role of abscisic acid (ABA), a recently discovered human inflammatory hormone, in silica-induced activation of murine RAW264.7 macrophages and of rat alveolar macrophages (AMs). Stimulation of both RAW264.7 cells and AMs with quartz induced a significant increase of ABA release (5- and 10-fold, respectively), compared to untreated cells. In RAW264.7 cells, autocrine ABA released after quartz stimulation sequentially activates the plasma membrane receptor LANCL2 and NADPH oxidase, generating a Ca(2+) influx resulting in NFκ B nuclear translocation and PGE(2) and TNF-α release (3-, 2-, and 3.5-fold increase, respectively, compared to control, unstimulated cells). Quartz-stimulated RAW264.7 cells silenced for LANCL2 or preincubated with a monoclonal antibody against ABA show an almost complete inhibition of NFκ B nuclear translocation and PGE(2) and TNF-α release compared to controls electroporated with a scramble oligonucleotide or preincubated with an unrelated antibody. AMs showed similar early and late ABA-induced responses as RAW264.7 cells. These findings identify ABA and LANCL2 as key mediators in quartz-induced inflammation, providing possible new targets for antisilicotic therapy. PMID:22042223

  16. CDK6 as a key regulator of hematopoietic and leukemic stem cell activation

    PubMed Central

    Scheicher, Ruth; Hoelbl-Kovacic, Andrea; Bellutti, Florian; Tigan, Anca-Sarmiza; Prchal-Murphy, Michaela; Heller, Gerwin; Schneckenleithner, Christine; Salazar-Roa, María; Zöchbauer-Müller, Sabine; Zuber, Johannes; Malumbres, Marcos; Kollmann, Karoline

    2015-01-01

    The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk6−/− HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more susceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcriptional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABLp210+ LSCs. Transplantation with BCR-ABLp210+–infected bone marrow from Cdk6−/− mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk6−/− BCR-ABLp210+ LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs. PMID:25342715

  17. Arctigenic acid, the key substance responsible for the hypoglycemic activity of Fructus Arctii.

    PubMed

    Xu, Zhaohui; Gu, Chenchen; Wang, Kai; Ju, Jiaxing; Wang, Haiying; Ruan, Kefeng; Feng, Yi

    2015-01-15

    We have reported the antidiabetic activity of the total lignans from Fructus arctii (TLFA) against alloxan-induced diabetes in mice and rats. In this study, arctigenic acid was found to be the main metabolite in rat plasma detected by UPLC/MS and HPLC/MS/MS after oral administration of TLFA. For the first time, its hypoglycemic activity and acute oral toxicity were evaluated in Goto-Kakizaki (GK) rats, a spontaneous type 2 diabetic animal model, and ICR mice respectively. GK rats were orally given arctigenic acid (50 mg/kg) twice daily before each meal for 12 weeks. The treatment reduced the elevated plasma glucose, glycosylated hemoglobin and showed significant improvement in glucose tolerance in glucose fed hyperglycemic GK rats. We found that the hypoglycemic effect of arctigenic acid was partly due to the stimulation on insulin secretion, whereas the body weight was not affected by arctigenic acid administration in GK rats. Meanwhile, there was no observable acute toxicity of arctigenic acid treatment at the dosage of 280 mg/kg body weight daily in the acute 14-day toxicity study in mice. This study demonstrates that arctigenic acid may be the main metabolite in the rat serum after oral administration of TLFA, which showed significant hypoglycemic effect in GK rats, and low acute toxicity in ICR mice. The result prompts us that arctigenic acid is the key substance responsible for Fructus Arctii antidiabetic activity and it has a great potential to be further developed as a novel therapeutic agent for diabetes in humans. PMID:25636881

  18. Geology is the Key to Explain Igneous Activity in the Mediterranean Area

    NASA Astrophysics Data System (ADS)

    Lustrino, M.

    2014-12-01

    Igneous activity in tectonically complex areas can be interpreted in many different ways, producing completely different petrogenetic models. Processes such as oceanic and continental subduction, lithospheric delamination, changes in subduction polarity, slab break-off and mantle plumes have all been advocated as causes for changes in plate boundaries and magma production, including rate and temporal distribution, in the circum-Mediterranean area. This region thus provides a natural laboratory to investigate a range of geodynamic and magmatic processes. Although many petrologic and tectonic models have been proposed, a number of highly controversial questions still remain. No consensus has yet been reached about the capacity of plate-tectonic processes to explain the origin and style of the magmatism. Similarly, there is still not consensus on the ability of geochemical and petrological arguments to reveal the geodynamic evolution of the area. The wide range of chemical and mineralogical magma compositions produced within and around the Mediterranean, from carbonatites to strongly silica-undersaturated silico-carbonatites and melilitites to strongly silica-oversaturated rhyolites, complicate models and usually require a large number of unconstrained assumptions. Can the calcalkaline-sodic alkaline transition be related to any common petrogenetic point? Is igneous activity plate-tectonic- (top-down) or deep-mantle-controlled (bottom-up)? Do the rare carbonatites and carbonate-rich igneous rocks derive from the deep mantle or a normal, CO2-bearing upper mantle? Do ultrapotassic compositions require continental subduction? Understanding chemically complex magmas emplaced in tectonically complex areas require open minds, and avoiding dogma and assumptions. Studying the geology and shallow dynamics, not speculating about the deep lower mantle, is the key to understanding the igneous activity.

  19. The active site architecture in peroxiredoxins: a case study on Mycobacterium tuberculosis AhpE.

    PubMed

    Pedre, Brandán; van Bergen, Laura A H; Palló, Anna; Rosado, Leonardo A; Dufe, Veronica Tamu; Molle, Inge Van; Wahni, Khadija; Erdogan, Huriye; Alonso, Mercedes; Proft, Frank De; Messens, Joris

    2016-08-11

    Peroxiredoxins catalyze the reduction of peroxides, a process of vital importance to survive oxidative stress. A nucleophilic cysteine, also known as the peroxidatic cysteine, is responsible for this catalytic process. We used the Mycobacterium tuberculosis alkyl hydroperoxide reductase E (MtAhpE) as a model to investigate the effect of the chemical environment on the specificity of the reaction. Using an integrative structural (R116A - PDB ; F37H - PDB ), kinetic and computational approach, we explain the mutational effects of key residues in its environment. This study shows that the active site residues are specifically oriented to create an environment which selectively favours a reaction with peroxides. PMID:27471753

  20. Targeting mechanisms at sites of complement activation for imaging and therapy.

    PubMed

    Holers, V Michael

    2016-06-01

    The complement system plays a key role in many acute injury states as well as chronic autoimmune and inflammatory diseases. Localized complement activation and alternative pathway-mediated amplification on diverse target surfaces promote local recruitment of pro-inflammatory cells and elaboration of other mediators. Despite a general understanding of the architecture of the system, though, many of the mechanisms that underlie site-specific complement activation and amplification in vivo are incompletely understood. In addition, there is no capability yet to measure the level of local tissue site-specific complement activation in patients without performing biopsies to detect products using immunohistochemical techniques. Herein is reviewed emerging evidence obtained through clinical research studies of human rheumatoid arthritis along with translational studies of its disease models which demonstrate that several parallel mechanisms are involved in site-specific amplification of activation of the complement system in vivo. Among these processes are de-regulation of the alternative pathway, effector pathway-catalyzed amplification of proximal complement activation, recognition of injury-associated ligands by components of the lectin pathway, and engagement of pathogenic natural antibodies that recognize a limited set of injury-associated neoepitopes. Studies suggest that each of these inter-related processes can play key roles in amplification of complement-dependent injury on self-tissues in vivo. These findings, in addition to development of an imaging strategy described herein designed to quantitatively measure local complement C3 fixation, have relevance to therapeutic and diagnostic strategies targeting the complement system. PMID:25979851

  1. Key amino acids of arabidopsis VKOR in the activity of phylloquinone reduction and disulfide bond formation.

    PubMed

    Yang, Xiao-Jian; Cui, Hao-Ran; Yu, Zhi-Bo; Du, Jia-Jia; Xu, Jia-Ning; Wang, Xiao-Yun

    2015-01-01

    Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process. The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved cysteines played crucial roles in the electron transfer process. PMID:25267254

  2. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  3. The Histamine N-Methyltransferase T105I Polymorphism Affects Active Site Structure and Dynamics†

    PubMed Central

    Rutherford, Karen; Parson, William W.; Daggett, Valerie

    2010-01-01

    Histamine N-methyltransferase (HNMT) is the sole enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels relative to the 105T protein. Crystal structures of both variants have been solved, but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations of both 105T and 105I at 37°C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened packing interactions between residue105 and residues within helix α3 and strand β3. This altered packing is directly translated to the active site resulting in the reorientation of several co-substrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lessens the hydrophobicity of the substrate-binding site. PMID:18154359

  4. Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

    PubMed Central

    Thangavelu, K.; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J.

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions. PMID:24451979

  5. Hydrogenosome Metabolism Is the Key Target for Antiparasitic Activity of Resveratrol against Trichomonas vaginalis

    PubMed Central

    Mallo, Natalia; Lamas, Jesús

    2013-01-01

    Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs. PMID:23478970

  6. Acid Is Key to the Radical-Trapping Antioxidant Activity of Nitroxides.

    PubMed

    Haidasz, Evan A; Meng, Derek; Amorati, Riccardo; Baschieri, Andrea; Ingold, Keith U; Valgimigli, Luca; Pratt, Derek A

    2016-04-27

    Persistent dialkylnitroxides (e.g., 2,2,6,6-tetramethylpiperidin-1-oxyl, TEMPO) play a central role in the activity of hindered amine light stabilizers (HALS)-additives that inhibit the (photo)oxidative degradation of consumer and industrial products. The accepted mechanism of HALS comprises a catalytic cycle involving the rapid combination of a nitroxide with an alkyl radical to yield an alkoxyamine that subsequently reacts with a peroxyl radical to eventually re-form the nitroxide. Herein, we offer evidence in favor of an alternative reaction mechanism involving the acid-catalyzed reaction of a nitroxide with a peroxyl radical to yield an oxoammonium ion followed by electron transfer from an alkyl radical to the oxoammonium ion to re-form the nitroxide. In preliminary work, we showed that TEMPO reacts with peroxyl radicals at diffusion-controlled rates in the presence of acids. Now, we show that TEMPO can be regenerated from its oxoammonium ion by reaction with alkyl radicals. We have determined that this reaction, which has been proposed to be a key step in TEMPO-catalyzed synthetic transformations, occurs with k ∼ 1-3 × 10(10) M(-1) s(-1), thereby enabling it to compete with O2 for alkyl radicals. The addition of weak acids facilitates this reaction, whereas the addition of strong acids slows it by enabling back electron transfer. The chemistry is shown to occur in hydrocarbon autoxidations at elevated temperatures without added acid due to the in situ formation of carboxylic acids, accounting for the long-known catalytic radical-trapping antioxidant activity of TEMPO that prompted the development of HALS. PMID:27023326

  7. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  8. Key Factors Influencing Rates of Heterotrophic Sulfate Reduction in Active Seafloor Hydrothermal Massive Sulfide Deposits

    PubMed Central

    Frank, Kiana L.; Rogers, Karyn L.; Rogers, Daniel R.; Johnston, David T.; Girguis, Peter R.

    2015-01-01

    Hydrothermal vents are thermally and geochemically dynamic habitats, and the organisms therein are subject to steep gradients in temperature and chemistry. To date, the influence of these environmental dynamics on microbial sulfate reduction has not been well constrained. Here, via multivariate experiments, we evaluate the effects of key environmental variables (temperature, pH, H2S, SO42−, DOC) on sulfate reduction rates and metabolic energy yields in material recovered from a hydrothermal flange from the Grotto edifice in the Main Endeavor Field, Juan de Fuca Ridge. Sulfate reduction was measured in batch reactions across a range of physico-chemical conditions. Temperature and pH were the strongest stimuli, and maximum sulfate reduction rates were observed at 50°C and pH 6, suggesting that the in situ community of sulfate-reducing organisms in Grotto flanges may be most active in a slightly acidic and moderate thermal/chemical regime. At pH 4, sulfate reduction rates increased with sulfide concentrations most likely due to the mitigation of metal toxicity. While substrate concentrations also influenced sulfate reduction rates, energy-rich conditions muted the effect of metabolic energetics on sulfate reduction rates. We posit that variability in sulfate reduction rates reflect the response of the active microbial consortia to environmental constraints on in situ microbial physiology, toxicity, and the type and extent of energy limitation. These experiments help to constrain models of the spatial contribution of heterotrophic sulfate reduction within the complex gradients inherent to seafloor hydrothermal deposits. PMID:26733984

  9. Key Factors Influencing Rates of Heterotrophic Sulfate Reduction in Active Seafloor Hydrothermal Massive Sulfide Deposits.

    PubMed

    Frank, Kiana L; Rogers, Karyn L; Rogers, Daniel R; Johnston, David T; Girguis, Peter R

    2015-01-01

    Hydrothermal vents are thermally and geochemically dynamic habitats, and the organisms therein are subject to steep gradients in temperature and chemistry. To date, the influence of these environmental dynamics on microbial sulfate reduction has not been well constrained. Here, via multivariate experiments, we evaluate the effects of key environmental variables (temperature, pH, H2S, [Formula: see text], DOC) on sulfate reduction rates and metabolic energy yields in material recovered from a hydrothermal flange from the Grotto edifice in the Main Endeavor Field, Juan de Fuca Ridge. Sulfate reduction was measured in batch reactions across a range of physico-chemical conditions. Temperature and pH were the strongest stimuli, and maximum sulfate reduction rates were observed at 50°C and pH 6, suggesting that the in situ community of sulfate-reducing organisms in Grotto flanges may be most active in a slightly acidic and moderate thermal/chemical regime. At pH 4, sulfate reduction rates increased with sulfide concentrations most likely due to the mitigation of metal toxicity. While substrate concentrations also influenced sulfate reduction rates, energy-rich conditions muted the effect of metabolic energetics on sulfate reduction rates. We posit that variability in sulfate reduction rates reflect the response of the active microbial consortia to environmental constraints on in situ microbial physiology, toxicity, and the type and extent of energy limitation. These experiments help to constrain models of the spatial contribution of heterotrophic sulfate reduction within the complex gradients inherent to seafloor hydrothermal deposits. PMID:26733984

  10. Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

    PubMed

    Irvine, Katherine L; Gangloff, Monique; Walsh, Catherine M; Spring, David R; Gay, Nicholas J; Bryant, Clare E

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  11. Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2

    PubMed Central

    Walsh, Catherine M.; Spring, David R.; Gay, Nicholas J.; Bryant, Clare E.

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  12. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  13. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    PubMed

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  14. Effect of urethane, dimethylnitrosamine, paraquat, and butylated hydroxytoluene on the activities of glycolytic key enzymes in mouse lung

    SciTech Connect

    Arany, I.; Rady, P.; Bojan, I.; Kertai, P.

    1981-12-01

    Effects of carcinogens and noncarcinogenic pulmonary toxicants on the activities of glycolytic key enzymes in the mouse lung were investigated. The carcinogens urethane (URTH) and dimethylnitrosamine (DMN) permanently enhanced, and the noncarcinogenic pulmonary toxicants paraquat (PAR) and butylated hydroxytoluene (BHT) temporarily, enhanced the activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the lungs of mice.

  15. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  16. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  17. Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation.

    PubMed

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-02-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate. PMID:23345443

  18. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  19. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  20. Analysis of the key active subsites of glycoside hydrolase 13 family members.

    PubMed

    Kumar, Vikash

    2010-05-01

    alpha-Amylase, pullulanase, neopullulanase, cyclomaltodextrinase (CDase), cyclomaltodextin glucanotransferase (CGTase), etc. are some of the amylolytic enzymes that act on polysaccharides. These enzymes differ from each other with respect to substrate and linkage specificities. These enzymes have been grouped into the GH13 (GH, Glycoside Hydrolase) family in the CAZy database on the basis of similarity in amino acid sequence. Members of this family share three domains viz., A, B, and C, which have several binding subsites to accommodate monomeric units of the polysaccharide substrate. Among these subsites, -2, -1, +1, and +2 subsites are the most critical subsites for catalytic activity. In the present study, the substrate analog-, inhibitor-, or product-bound 3-D structures of 24 members of GH13 family have been analyzed to identify the features of the -2, -1, +1, and +2 subsites shared by all the members for recognition of the common substrate. It is found that neither the number nor the nature of the potential hydrogen bond-forming residues is conserved with the exception of the presence of tyrosine as a stacking residue in the -1 subsite. The relative spatial disposition of the conserved subsite residues are conserved as judged by distance matrices. The backbone of the -2, -1, +1, and +2 subsites does not undergo conformational change for the recognition of the substrate. This analysis suggests that these enzymes recognize their substrate on the basis of shape of the substrate rather than on the basis of specific interactions within the binding site. PMID:20227065

  1. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

  2. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  3. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

  4. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  5. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  6. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  7. Active site and laminarin binding in glycoside hydrolase family 55.

    PubMed

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  8. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  9. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    PubMed Central

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  10. Calcitonin gene-related peptide is a key factor in the homing of transplanted human MSCs to sites of spinal cord injury.

    PubMed

    Zhang, Yu; Yang, Jinhua; Zhang, Peng; Liu, Tao; Xu, Jianwei; Fan, Zhihai; Shen, Yixin; Li, Wenjie; Zhang, Huanxiang

    2016-01-01

    Mesenchymal stem cells (MSCs) can be used to treat many diseases, including spinal cord injury (SCI). Treatment relies mostly on the precise navigation of cells to the injury site for rebuilding the damaged spinal cord. However, the key factors guiding MSCs to the epicenter of SCI remain unknown. Here, we demonstrated that calcitonin gene-related peptide (CGRP), a neural peptide synthesized in spinal cord, can dramatically aid the homing of human umbilical cord mesenchymal stem cells (HUMSCs) in spinal cord-transected SCI rats. First, HUMSCs exhibited chemotactic responses in vitro to CGRP. By time-lapse video analysis, increased chemotactic index (CMI), forward migration index (FMI) and speed contributed to this observed migration. Then, through enzyme immunoassay, higher CGRP concentrations at the lesion site were observed after injury. The release of CGRP directed HUMSCs to the injury site, which was suppressed by CGRP 8-37, a CGRP antagonist. We also verified that the PI3K/Akt and p38MAPK signaling pathways played a critical role in the CGRP-induced chemotactic migration of HUMSCs. Collectively, our data reveal that CGRP is a key chemokine that helps HUMSCs migrate to the lesion site and thereby can be used as a model molecule to study MSCs homing after SCI. PMID:27296555

  11. Calcitonin gene-related peptide is a key factor in the homing of transplanted human MSCs to sites of spinal cord injury

    PubMed Central

    Zhang, Yu; Yang, Jinhua; Zhang, Peng; Liu, Tao; Xu, Jianwei; Fan, Zhihai; Shen, Yixin; Li, Wenjie; Zhang, Huanxiang

    2016-01-01

    Mesenchymal stem cells (MSCs) can be used to treat many diseases, including spinal cord injury (SCI). Treatment relies mostly on the precise navigation of cells to the injury site for rebuilding the damaged spinal cord. However, the key factors guiding MSCs to the epicenter of SCI remain unknown. Here, we demonstrated that calcitonin gene-related peptide (CGRP), a neural peptide synthesized in spinal cord, can dramatically aid the homing of human umbilical cord mesenchymal stem cells (HUMSCs) in spinal cord-transected SCI rats. First, HUMSCs exhibited chemotactic responses in vitro to CGRP. By time-lapse video analysis, increased chemotactic index (CMI), forward migration index (FMI) and speed contributed to this observed migration. Then, through enzyme immunoassay, higher CGRP concentrations at the lesion site were observed after injury. The release of CGRP directed HUMSCs to the injury site, which was suppressed by CGRP 8–37, a CGRP antagonist. We also verified that the PI3K/Akt and p38MAPK signaling pathways played a critical role in the CGRP-induced chemotactic migration of HUMSCs. Collectively, our data reveal that CGRP is a key chemokine that helps HUMSCs migrate to the lesion site and thereby can be used as a model molecule to study MSCs homing after SCI. PMID:27296555

  12. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  13. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions. PMID:25555911

  14. Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

    PubMed Central

    Sigala, Paul A.; Fafarman, Aaron T.; Schwans, Jason P.; Fried, Stephen D.; Fenn, Timothy D.; Caaveiro, Jose M. M.; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A.; Boxer, Steven G.; Herschlag, Daniel

    2013-01-01

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

  15. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  16. Representing Microbial Dormancy in Soil Decomposition Models Improves Model Performance and Reveals Key Ecosystem Controls on Microbial Activity

    NASA Astrophysics Data System (ADS)

    He, Y.; Yang, J.; Zhuang, Q.; Wang, G.; Liu, Y.

    2014-12-01

    Climate feedbacks from soils can result from environmental change and subsequent responses of plant and microbial communities and nutrient cycling. Explicit consideration of microbial life history traits and strategy may be necessary to predict climate feedbacks due to microbial physiology and community changes and their associated effect on carbon cycling. In this study, we developed an explicit microbial-enzyme decomposition model and examined model performance with and without representation of dormancy at six temperate forest sites with observed soil efflux ranged from 4 to 10 years across different forest types. We then extrapolated the model to all temperate forests in the Northern Hemisphere (25-50°N) to investigate spatial controls on microbial and soil C dynamics. Both models captured the observed soil heterotrophic respiration (RH), yet no-dormancy model consistently exhibited large seasonal amplitude and overestimation in microbial biomass. Spatially, the total RH from temperate forests based on dormancy model amounts to 6.88PgC/yr, and 7.99PgC/yr based on no-dormancy model. However, no-dormancy model notably overestimated the ratio of microbial biomass to SOC. Spatial correlation analysis revealed key controls of soil C:N ratio on the active proportion of microbial biomass, whereas local dormancy is primarily controlled by soil moisture and temperature, indicating scale-dependent environmental and biotic controls on microbial and SOC dynamics. These developments should provide essential support to modeling future soil carbon dynamics and enhance the avenue for collaboration between empirical soil experiment and modeling in the sense that more microbial physiological measurements are needed to better constrain and evaluate the models.

  17. Car-Parrinello study of Ziegler-Natta heterogeneous catalysis: stability and destabilization problems of the active site models

    NASA Astrophysics Data System (ADS)

    Boero, Mauro; Parrinello, Michele; Terakura, Kiyoyuki; Weiss, Horst

    Recent results on Car-Parrinello simulations of MgCl 2 /TiCl 4 Ziegler-Natta heterogeneous catalytic systems are reviewed and the stability of Ti active site configurations on the various active surfaces are discussed. In particular, the focus is on the ability of an active centre to carry out the polymerization process efficiently, achieved by monitoring geometrical changes and associated energetics during the reaction. The active site geometry is crucial in enhancing or decreasing the activation barrier, in selecting the olefin enantioface and in some cases in destabilizing the catalytic centre. A key point in the binding/destabilization interplay is the matching of Ti with the substrate, an issue that represents a fundamental difference with respect to homogeneous systems.

  18. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  19. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  20. California Energy Incentive Programs: An Annual Update on Key Energy Issues and Financial Opportunities for Federal Sites in California

    SciTech Connect

    2011-12-01

    A spate of recently enacted energy legislation and associated program changes is providing numerous opportunities to help California federal energy managers cut costs and meet their renewables, energy efficiency and GHG emissions goals. In April 2011, Governor Jerry Brown approved the nation’s most ambitious renewable portfolio standard (RPS), which requires 33% of the state’s electricity to come from renewable energy sources by 2020. Policy changes that will support the RPS include expanded eligibility rules that fill previous gaps in incentives for certain sizes of on-site renewable energy systems. Program updates described in this document include: $200 million more in funding for California Solar Initiative rebates to commercial and industrial customers; an increase in the eligible system size for the Feed-In-Tariff (FIT) from 1.5MW to 3MW; and pending changes that may allow customer-side systems to sell tradable renewable energy credits (TRECs) to entities with RPS compliance obligations in California.

  1. Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys

    PubMed Central

    Spade, Daniel J.; Griffitt, Robert J.; Liu, Li; Brown-Peterson, Nancy J.; Kroll, Kevin J.; Feswick, April; Glazer, Robert A.; Barber, David S.; Denslow, Nancy D.

    2010-01-01

    Background Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. Methodology/Principal Findings Histology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. Conclusions/Significance Congruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34

  2. Long-term monitoring of soil microbiological activities in two forest sites in South tyrol in the italian alps.

    PubMed

    Margesin, Rosa; Minerbi, Stefano; Schinner, Franz

    2014-09-17

    We monitored microbiological properties in two forest sites over a period of 17 years (1993-2010) within the International Cooperative Programme on Integrated Monitoring of Air Pollution Effects on Ecosystems (ICP IM). The two study sites were located in South Tyrol in the Italian Alps at altitudes of 1,737 m a.s.l. (subalpine site IT01) and 570 m a.s.l. (submontane site IT02). Soil samples were collected in the late spring and autumn of 1993, 2000, and 2010, and were characterized by measuring respiration, key enzyme activities involved in the C, N, P, and S cycles and litter degradation, and the abundance of viable bacterial and fungal populations. Over the study period, an increase in mean annual air temperatures at both sites (+0.6°C and +0.8°C at IT01 and IT02, respectively) was calculated from trendlines. Significantly lower mean annual air temperatures, higher temperature fluctuations, and higher annual precipitation rates were observed at site IT01 than at site IT02. Subalpine site IT01 was characterized by significantly lower microbial activity (respiration, enzymes) and abundance than those at submontane site IT02. The year of sampling had a significant effect on all the parameters investigated, except for nitrification. Fungal abundance decreased consistently over the study period, while no consistent trend was noted among the other parameters investigated. Season only affected a few of the measured microbiological parameters: respiration and bacterial numbers were significantly higher in the spring than in the autumn, while the opposite was noted for xylanase and phosphatase activities. Soil fungi contributed essentially to xylanase and protease activities, while soil bacteria were mainly involved in degradation processes that required the activity of sulfatase. PMID:25008018

  3. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  4. Experiment Dashboard - a generic, scalable solution for monitoring of the LHC computing activities, distributed sites and services

    NASA Astrophysics Data System (ADS)

    Andreeva, J.; Cinquilli, M.; Dieguez, D.; Dzhunov, I.; Karavakis, E.; Karhula, P.; Kenyon, M.; Kokoszkiewicz, L.; Nowotka, M.; Ro, G.; Saiz, P.; Sargsyan, L.; Schovancova, J.; Tuckett, D.

    2012-12-01

    The Experiment Dashboard system provides common solutions for monitoring job processing, data transfers and site/service usability. Over the last seven years, it proved to play a crucial role in the monitoring of the LHC computing activities, distributed sites and services. It has been one of the key elements during the commissioning of the distributed computing systems of the LHC experiments. The first years of data taking represented a serious test for Experiment Dashboard in terms of functionality, scalability and performance. And given that the usage of the Experiment Dashboard applications has been steadily increasing over time, it can be asserted that all the objectives were fully accomplished.

  5. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase.

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

    Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. PMID:16185065

  6. Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator

    PubMed Central

    Kitatsuji, Chihiro; Izumi, Kozue; Nambu, Shusuke; Kurogochi, Masaki; Uchida, Takeshi; Nishimura, Shin-Ichiro; Iwai, Kazuhiro; O’Brian, Mark R.; Ikeda-Saito, Masao; Ishimori, Koichiro

    2016-01-01

    The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the “active site conversion” from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel “two-step self-oxidative modification” mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion. PMID:26729068

  7. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  8. Photoreduction of Hg(ii) and photodemethylation of methylmercury: the key role of thiol sites on dissolved organic matter.

    PubMed

    Jeremiason, Jeffrey D; Portner, Joshua C; Aiken, George R; Hiranaka, Amber J; Dvorak, Michelle T; Tran, Khuyen T; Latch, Douglas E

    2015-11-01

    This study examined the kinetics of photoreduction of Hg(ii) and photodemethylation of methylmercury (MeHg(+)) attached to, or in the presence of, dissolved organic matter (DOM). Both Hg(ii) and MeHg(+) are principally bound to reduced sulfur groups associated with DOM in many freshwater systems. We propose that a direct photolysis mechanism is plausible for reduction of Hg(ii) bound to reduced sulfur groups on DOM while an indirect mechanism is supported for photodemethylation of MeHg(+) bound to DOM. UV spectra of Hg(ii) and MeHg(+) bound to thiol containing molecules demonstrate that the Hg(ii)-S bond is capable of absorbing UV-light in the solar spectrum to a much greater extent than MeHg(+)-S bonds. Experiments with chemically distinct DOM isolates suggest that concentration of DOM matters little in the photochemistry if there are enough reduced S sites present to strongly bind MeHg(+) and Hg(ii); DOM concentration does not play a prominent role in photodemethylation other than to screen light, which was demonstrated in a field experiment in the highly colored St. Louis River where photodemethylation was not observed at depths ≥ 10 cm. Experiments with thiol ligands yielded slower photodegradation rates for MeHg(+) than in experiments with DOM and thiols; rates in the presence of DOM alone were the fastest supporting an intra-DOM mechanism. Hg(ii) photoreduction rates, however, were similar in experiments with only DOM, thiols plus DOM, or only thiols suggesting a direct photolysis mechanism. Quenching experiments also support the existence of an intra-DOM photodemethylation mechanism for MeHg(+). Utilizing the difference in photodemethylation rates measured for MeHg(+) attached to DOM or thiol ligands, the binding constant for MeHg(+) attached to thiol groups on DOM was estimated to be 10(16.7). PMID:26420634

  9. Mutation of conserved active-site threonine residues in creatine kinase affects autophosphorylation and enzyme kinetics.

    PubMed Central

    Stolz, Martin; Hornemann, Thorsten; Schlattner, Uwe; Wallimann, Theo

    2002-01-01

    Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics. It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier [Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim. Biophys. Acta 1251, 81-90], or exclusively nucleotidylated. In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated. However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels. Using phosphoamino-acid analysis of (32)P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes. The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified. Site-directed threonine-valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues. The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself. PMID:11964180

  10. Comparative studies on the inhibitory activities of selected benzoic acid derivatives against secretory phospholipase A2, a key enzyme involved in the inflammatory pathway.

    PubMed

    Dileep, K V; Remya, C; Cerezo, J; Fassihi, A; Pérez-Sánchez, H; Sadasivan, C

    2015-07-01

    Inflammation is considered to be a key factor in major diseases like cancer, Alzheimer's disease, Parkinson's disease, etc. For the past few decades, pharmaceutical companies have explored new effective medications against inflammation. As a part of their detailed studies, many drug targets and drugs have been introduced against inflammation. In the present study, the inhibiting capacities of selected benzoic acid derivatives like gallic acid, vannilic acid, syringic acid and protocatechuic acid against secretory phospholipase A2 (sPLA2), a major enzyme involved in the inflammatory pathway, have been investigated. The detailed in vitro, biophysical and in silico studies carried out on these benzoic acid derivatives revealed that all the selected compounds have a uniform mode of binding in the active site of sPLA2 and are inhibitory in micromolar concentrations. The study also focuses on the non-selective inhibitory activity of an NSAID, aspirin, against sPLA2. PMID:25927625

  11. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  12. Construction of DNA recognition sites active in Haemophilus transformation.

    PubMed Central

    Danner, D B; Smith, H O; Narang, S A

    1982-01-01

    Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer. Images PMID:6285382

  13. Characterization of active site residues of nitroalkane oxidase.

    PubMed

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

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  14. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  15. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  16. Acrolein Modification Impairs Key Functional Features of Rat Apolipoprotein E: Identification of Modified Sites by Mass Spectrometry

    PubMed Central

    Tran, Tuyen N.; Kosaraju, Malathi G.; Tamamizu-Kato, Shiori; Akintunde, Olayemi; Zheng, Ying; Bielicki, John K.; Pinkerton, Kent; Uchida, Koji; Lee, Yuan Yu; Narayanaswami, Vasanthy

    2014-01-01

    Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for low-density lipoprotein receptor (LDLr) family of proteins, in addition to playing a role in promoting macrophage cholesterol efflux in atherosclerotic lesions. The objective of this study is to examine the effect of acrolein modification on the structure and function of rat apoE and to determine sites and nature of modification by mass spectrometry. Acrolein is a highly reactive aldehyde, which is generated endogenously as one of the products of lipid peroxidation and is present in the environment in pollutants such as tobacco smoke and heated oils. In initial studies, acrolein-modified apoE was identified by immunoprecipitation using an acrolein-lysine specific antibody, in the plasma of ten-week old male rats that were exposed to filtered air (FA) or low doses of environmental tobacco smoke (ETS). While both groups displayed acrolein-modified apoE in the lipoprotein fraction, the ETS group had higher levels in lipid-free fraction compared to the FA group. This observation provided the rationale to further investigate the effect of acrolein modification on rat apoE at a molecular level. Treatment of recombinant rat apoE with a 10-fold molar excess of acrolein resulted in: (i) a significant decrease in lipid-binding and cholesterol efflux abilities, (ii) impairment in the LDLr- and heparin-binding capabilities, and (iii) significant alterations in the overall stability of the protein. The disruption in the functional abilities is attributed directly or indirectly to acrolein modification yielding: an aldimine adduct at K149 and K155 (+38); a propanal adduct at K135 and K138 (+56); an Nε-(3-methylpyridinium)lysine (MP-lysine) at K64, K67 and K254 (+76), and Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) derivative at position K68 (+94), as determined by Matrix-Assisted Laser

  17. Active polarization stabilization in optical fibers suitable for quantum key distribution.

    PubMed

    Chen, Jie; Wu, Guang; Li, Yao; Wu, E; Zeng, Heping

    2007-12-24

    Polarization feedback control of single-photon pulses has been achieved in long-distance fibers for more than 10 hours, which facilitated "one-way" polarization-encoded quantum key distribution with long-term stabilities. Experimental test of polarization encoding in 75 km fibers demonstrated that the single-photon polarization transformation in long-distance fibers could be controlled to provide a typical QBER of (3.9+/-1.5)% within a long-term operation of 620 minutes. PMID:19551088

  18. NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction

    PubMed Central

    Katkar, Gajanan D.; Sundaram, Mahalingam S.; NaveenKumar, Somanathapura K.; Swethakumar, Basavarajaiah; Sharma, Rachana D.; Paul, Manoj; Vishalakshi, Gopalapura J.; Devaraja, Sannaningaiah; Girish, Kesturu S.; Kemparaju, Kempaiah

    2016-01-01

    Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom. PMID:27093631

  19. NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction.

    PubMed

    Katkar, Gajanan D; Sundaram, Mahalingam S; NaveenKumar, Somanathapura K; Swethakumar, Basavarajaiah; Sharma, Rachana D; Paul, Manoj; Vishalakshi, Gopalapura J; Devaraja, Sannaningaiah; Girish, Kesturu S; Kemparaju, Kempaiah

    2016-01-01

    Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom. PMID:27093631

  20. Physical Activity in Child-Care Centers: Do Teachers Hold the Key to the Playground?

    ERIC Educational Resources Information Center

    Copeland, Kristen A.; Kendeigh, Cassandra A.; Saelens, Brian E.; Kalkwarf, Heidi J.; Sherman, Susan N.

    2012-01-01

    Many (56%) US children aged 3-5 years are in center-based childcare and are not obtaining recommended levels of physical activity. In order to determine what child-care teachers/providers perceived as benefits and barriers to children's physical activity in child-care centers, we conducted nine focus groups and 13 one-on-one interviews with 49…

  1. An Activity-Based Learning Approach for Key Geographical Information Systems (GIS) Concepts

    ERIC Educational Resources Information Center

    Srivastava, Sanjeev Kumar; Tait, Cynthia

    2012-01-01

    This study presents the effect of active learning methods of concepts in geographical information systems where students participated in a series of interlocked learning experiences. These activities spanned several teaching weeks and involved the creation of a hand drawn map that was scanned and geo-referenced with locations' coordinates derived…

  2. Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.

    PubMed

    Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E; Khosla, Chaitan

    2016-08-16

    Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs. PMID:27441852

  3. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  4. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  5. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  6. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  7. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  8. Catalytic roles of flexible regions at the active site of ribulose-bisphosphate carboxylase/oxygenase (Rubisco)

    SciTech Connect

    Hartman, F.C.; Harpel, M.R.; Chen, Yuh-Ru; Larson, E.M.; Larimer, F.W.

    1995-12-31

    Chemical and mutagenesis studies of Rubisco have identified Lys329 and Glu48 as active-site residues that are located in distinct, interacting domains from adjacent subunits. Crystallographic analyses have shown that Lys329 is the apical residue in a 12-residue flexible loop (loop 6) of the {Beta},{alpha}-barrel domain of the active site and that Glu48 resides at the end of helix B of the N-terminal domain of the active site. When phosphorylated ligands are bound by the enzyme, loop 6 adopts a closed conformation and, in concert with repositioning of helix B, thereby occludes the active site from the external environment. In this closed conformation, the {gamma}-carboxylate of Glu48 and the {epsilon}-amino group of Lys329 engage in intersubunit electrostatic interaction. By use of appropriate site-directed mutants of Rhodospirillum rubrum Rubisco, we are addressing several issues: the catalytic roles of Lys329 and Glu48, the functional significance of the intersubunit salt bridge comprised of these two residues, and the roles of loop 6 and helix B in stabilizing labile reaction intermediates. Characterization of novel products derived from misprocessing of D-ribulose-1,5-bisphosphate (RuBP) by the mutant proteins have illuminated the structure of the key intermediate in the normal oxygenase pathway.

  9. Dynamic Transcription Factor Activity Profiles Reveal Key Regulatory Interactions During Megakaryocytic and Erythroid Differentiation

    PubMed Central

    Duncan, Mark T.; Shin, Seungjin; Wu, Jia J.; Mays, Zachary; Weng, Stanley; Bagheri, Neda; Miller, William M.; Shea, Lonnie D.

    2014-01-01

    The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. PMID:24853077

  10. Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation.

    PubMed

    Duncan, Mark T; Shin, Seungjin; Wu, Jia J; Mays, Zachary; Weng, Stanley; Bagheri, Neda; Miller, William M; Shea, Lonnie D

    2014-10-01

    The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. PMID:24853077

  11. Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.

    PubMed

    Rossetto, O; Caccin, P; Rigoni, M; Tonello, F; Bortoletto, N; Stevens, R C; Montecucco, C

    2001-08-01

    Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT. PMID:11306125

  12. Transcriptomic Analysis of Musca domestica to Reveal Key Genes of the Prophenoloxidase-Activating System

    PubMed Central

    Li, Dianxiang; Liang, Yongli; Wang, Xianwei; Wang, Lei; Qi, Mei; Yu, Yang; Luan, Yuanyuan

    2015-01-01

    The proPO system regulates melanization in arthropods. However, the genes that are involved in the proPO system in housefly Musca domestica remain unclear. Thus, this study analyzed the combined transcriptome obtained from M. domestica larvae, pupae, and adults that were either normal or bacteria-challenged by an Escherichia coli and Staphylococcus aureus mixture. A total of 54,821,138 clean reads (4.93 Gb) were yielded by Illumina sequencing, which were de novo assembled into 89,842 unigenes. Of the 89,842 unigenes, based on a similarity search with known genes in other insects, 24 putative genes related to the proPO system were identified. Eight of the identified genes encoded for peptidoglycan recognition receptors, two encoded for prophenoloxidases, three encoded for prophenoloxidase-activating enzymes, and 11 encoded for serine proteinase inhibitors. The expression levels of these identified genes were investigated by qRT-PCR assay, which were consistent with expected activation process of the proPO system, and their activation functions were confirmed by the measurement of phenoloxidase activity in bacteria-infected larvae after proPO antibody blockage, suggesting these candidate genes might have potentially different roles in the activation of proPO system. Collectively, this study has provided the comprehensive transcriptomic data of an insect and some fundamental basis toward achieving understanding of the activation mechanisms and immune functions of the proPO system in M. domestica. PMID:26156588

  13. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  14. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

    Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

  15. Prediction of Geomagnetic Activity and Key Parameters in High-latitude Ionosphere

    NASA Technical Reports Server (NTRS)

    Khazanov, George V.; Lyatsky, Wladislaw; Tan, Arjun; Ridley, Aaron

    2007-01-01

    Prediction of geomagnetic activity and related events in the Earth's magnetosphere and ionosphere are important tasks of US Space Weather Program. Prediction reliability is dependent on the prediction method, and elements included in the prediction scheme. Two of the main elements of such prediction scheme are: an appropriate geomagnetic activity index, and an appropriate coupling function (the combination of solar wind parameters providing the best correlation between upstream solar wind data and geomagnetic activity). We have developed a new index of geomagnetic activity, the Polar Magnetic (PM) index and an improved version of solar wind coupling function. PM index is similar to the existing polar cap PC index but it shows much better correlation with upstream solar wind/IMF data and other events in the magnetosphere and ionosphere. We investigate the correlation of PM index with upstream solar wind/IMF data for 10 years (1995-2004) that include both low and high solar activity. We also have introduced a new prediction function for the predicting of cross-polar-cap voltage and Joule heating based on using both PM index and upstream solar wind/IMF data. As we show such prediction function significantly increase the reliability of prediction of these important parameters. The correlation coefficients between the actual and predicted values of these parameters are approx. 0.9 and higher.

  16. Key amino acid residues for the endo-processive activity of GH74 xyloglucanase.

    PubMed

    Matsuzawa, Tomohiko; Saito, Yuji; Yaoi, Katsuro

    2014-05-01

    Unlike endo-dissociative-xyloglucanases, Paenibacillus XEG74 is an endo-processive xyloglucanase that contains four unique tryptophan residues in the negative subsites (W61 and W64) and the positive subsites (W318 and W319), as indicated by three-dimensional homology modelling. Selective replacement of the positive subsite residues with alanine mutations reduced the degree of processive activity and resulted in the more endo-dissociative-activity. The results showed that W318 and W319, which are found in the positive subsites, are essential for processive degradation and are responsible for maintaining binding interactions with xyloglucan polysaccharide through a stacking effect. PMID:24657616

  17. Key structure-activity relationships in the vanadium phosphorus oxide catalyst system

    SciTech Connect

    Thompson, M.R. ); Ebner, J.R. )

    1990-04-01

    The crystal structure of vanadyl pyrophosphate has been redetermined using single crystals obtained from a near solidified melt of a microcrystalline catalyst sample. Crystals that index as vanadyl pyrophosphate obtained from this melt are variable in color. Crystallographic refinement of the single crystal x-ray diffraction data indicates that structural differences among these materials can be described in terms of crystal defects associated with linear disorder of the vanadium atoms. The importance of the disorder is outlined in the context of its effect on the proposed surface topology parallel to (1,0,0). Models of the surface topology simply and intuitively account for the non-stoichometric surface atomic P/V ratio exhibited by selective catalysts of this phase. These models also point to the possible role of the excess phosphorus in providing site isolation of reactive centers at the surface. 33 refs., 7 figs.

  18. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    SciTech Connect

    Teese, G.D.

    1995-09-28

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers.

  19. Endocrine disrupting activities of surface water associated with a West Virginia oil and gas industry wastewater disposal site

    USGS Publications Warehouse

    Kassotis, Christopher D.; Iwanowicz, Luke; Akob, Denise M.; Cozzarelli, Isabelle M.; Mumford, Adam; Orem, William H.; Nagel, Susan C.

    2016-01-01

    Currently, >95% of end disposal of hydraulic fracturing wastewater from unconventional oil and gas operations in the US occurs via injection wells. Key data gaps exist in understanding the potential impact of underground injection on surface water quality and environmental health. The goal of this study was to assess endocrine disrupting activity in surface water at a West Virginia injection well disposal site. Water samples were collected from a background site in the area and upstream, on, and downstream of the disposal facility. Samples were solid-phase extracted, and extracts assessed for agonist and antagonist hormonal activities for five hormone receptors in mammalian and yeast reporter gene assays. Compared to reference water extracts upstream and distal to the disposal well, samples collected adjacent and downstream exhibited considerably higher antagonist activity for the estrogen, androgen, progesterone, glucocorticoid and thyroid hormone receptors. In contrast, low levels of agonist activity were measured in upstream/distal sites, and were inhibited or absent at downstream sites with significant antagonism. Concurrent analyses by partner laboratories (published separately) describe the analytical and geochemical profiling of the water; elevated conductivity as well as high sodium, chloride, strontium, and barium concentrations indicate impacts due to handling of unconventional oil and gas wastewater. Notably, antagonist activities in downstream samples were at equivalent authentic standard concentrations known to disrupt reproduction and/or development in aquatic animals. Given the widespread use of injection wells for end-disposal of hydraulic fracturing wastewater, these data raise concerns for human and animal health nearby.

  20. Endocrine disrupting activities of surface water associated with a West Virginia oil and gas industry wastewater disposal site.

    PubMed

    Kassotis, Christopher D; Iwanowicz, Luke R; Akob, Denise M; Cozzarelli, Isabelle M; Mumford, Adam C; Orem, William H; Nagel, Susan C

    2016-07-01

    Currently, >95% of end disposal of hydraulic fracturing wastewater from unconventional oil and gas operations in the US occurs via injection wells. Key data gaps exist in understanding the potential impact of underground injection on surface water quality and environmental health. The goal of this study was to assess endocrine disrupting activity in surface water at a West Virginia injection well disposal site. Water samples were collected from a background site in the area and upstream, on, and downstream of the disposal facility. Samples were solid-phase extracted, and extracts assessed for agonist and antagonist hormonal activities for five hormone receptors in mammalian and yeast reporter gene assays. Compared to reference water extracts upstream and distal to the disposal well, samples collected adjacent and downstream exhibited considerably higher antagonist activity for the estrogen, androgen, progesterone, glucocorticoid and thyroid hormone receptors. In contrast, low levels of agonist activity were measured in upstream/distal sites, and were inhibited or absent at downstream sites with significant antagonism. Concurrent analyses by partner laboratories (published separately) describe the analytical and geochemical profiling of the water; elevated conductivity as well as high sodium, chloride, strontium, and barium concentrations indicate impacts due to handling of unconventional oil and gas wastewater. Notably, antagonist activities in downstream samples were at equivalent authentic standard concentrations known to disrupt reproduction and/or development in aquatic animals. Given the widespread use of injection wells for end-disposal of hydraulic fracturing wastewater, these data raise concerns for human and animal health nearby. PMID:27073166

  1. Florida Keys

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The Florida Keys are a chain of islands, islets and reefs extending from Virginia Key to the Dry Tortugas for about 309 kilometers (192 miles). The keys are chiefly limestone and coral formations. The larger islands of the group are Key West (with its airport), Key Largo, Sugarloaf Key, and Boca Chica Key. A causeway extends from the mainland to Key West.

    This image was acquired on October 28, 2001, by the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) on NASA's Terra satellite. With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER images Earth to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18, 1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products.

    The broad spectral coverage and high spectral resolution of ASTER will provide scientists in numerous disciplines with critical information for surface mapping, and monitoring of dynamic conditions and temporal change. Example applications are: monitoring glacial advances and retreats; monitoring potentially active volcanoes; identifying crop stress; determining cloud morphology and physical properties; wetlands evaluation; thermal pollution monitoring; coral reef degradation; surface temperature mapping of soils and geology; and measuring surface heat balance.

    Dr. Anne Kahle at NASA's Jet Propulsion Laboratory, Pasadena, Calif., is the U.S. Science team leader; Bjorn Eng of JPL is the project manager. The Terra mission is part of NASA's Earth Science Enterprise, a long- term research effort to understand and protect our home planet. Through the study of Earth, NASA will help to provide sound science to policy and economic

  2. Collaboration with aviation — The key to commercialisation of space activities

    NASA Astrophysics Data System (ADS)

    Collins, Patrick; Funatsu, Yoshiyuki

    2000-07-01

    The US government's Commercial Space Act of 1998 and commitment to commercialise the International Space Station's operations have changed the direction of space development in the post-cold-war world definitively. During 1998 also the feasibility and great economic potential of space travel by the general public was acknowledged in publications by NASA, AIAA and the Japanese Keidanren. However, crewed space activities are all taxpayer-funded, primarily for scientific research; they have involved only a few hundred people traveling to space to date; and those involved have no experience of commercial passenger service operations. By contrast, aviation is a global industry, largely commercial, involving the range of activities from engineering design to marketing, and serving more than 1 billion passengers/year. Aviation has very high safety levels developed over decades of experience of carrying billions of passengers. Furthermore, the aviation industry also has extensive experience of operating rocket-powered piloted vehicles: during the 1950s several countries operated such vehicles sufficiently frequently to develop routine operations, maintenance and repair procedures. Consequently, in order to develop safe and profitable passenger travel services to, from and in space, people, companies and organisations with experience of space activities have a great deal to gain from collaboration with all parts of the aviation industry. Due to the potential economic value of this development, and the high cost to taxpayers of space activities today, governments should take steps to start this collaboration as soon as possible.

  3. Physical activity in child-care centers: do teachers hold the key to the playground?

    PubMed Central

    Copeland, Kristen A.; Kendeigh, Cassandra A.; Saelens, Brian E.; Kalkwarf, Heidi J.; Sherman, Susan N.

    2012-01-01

    Many (56%) US children aged 3–5 years are in center-based childcare and are not obtaining recommended levels of physical activity. In order to determine what child-care teachers/providers perceived as benefits and barriers to children’s physical activity in child-care centers, we conducted nine focus groups and 13 one-on-one interviews with 49 child-care teachers/providers in Cincinnati, OH. Participants noted physical and socio-emotional benefits of physical activity particular to preschoolers (e.g. gross motor skill development, self-confidence after mastery of new skills and improved mood, attention and napping after exercise) but also noted several barriers including their own personal attitudes (e.g. low self-efficacy) and preferences to avoid the outdoors (e.g. don’t like hot/cold weather, getting dirty, chaos of playground). Because individual teachers determine daily schedules and ultimately make the decision whether to take the children outdoors, they serve as gatekeepers to the playground. Participants discussed a spectrum of roles on the playground, from facilitator to chaperone to physical activity inhibitor. These findings suggest that children could have very different gross motor experiences even within the same facility (with presumably the same environment and policies), based on the beliefs, creativity and level of engagement of their teacher. PMID:21804083

  4. The Key Factors of an Active Learning Method in a Microprocessors Course

    ERIC Educational Resources Information Center

    Carpeno, A.; Arriaga, J.; Corredor, J.; Hernandez, J.

    2011-01-01

    The creation of the European Higher Education Area (EHEA) is promoting a change toward a new model of education focused on the student. It is impelling methodological innovation processes in many European universities, leading more teachers to apply methods based on active and cooperative learning in their classrooms. However, the successful…

  5. Behavioral science in video games for children's diet and physical activity change: Key research needs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Innovative intervention programs are needed to overcome the limitations in previous programs that promoted change in diabetes risk behaviors in children. Serious video games show promise of changing dietary and physical activity behaviors, but research is needed on the optimal design of behavior-cha...

  6. Physical activity in child-care centers: do teachers hold the key to the playground?

    PubMed

    Copeland, Kristen A; Kendeigh, Cassandra A; Saelens, Brian E; Kalkwarf, Heidi J; Sherman, Susan N

    2012-02-01

    Many (56%) US children aged 3-5 years are in center-based childcare and are not obtaining recommended levels of physical activity. In order to determine what child-care teachers/providers perceived as benefits and barriers to children's physical activity in child-care centers, we conducted nine focus groups and 13 one-on-one interviews with 49 child-care teachers/providers in Cincinnati, OH. Participants noted physical and socio-emotional benefits of physical activity particular to preschoolers (e.g. gross motor skill development, self-confidence after mastery of new skills and improved mood, attention and napping after exercise) but also noted several barriers including their own personal attitudes (e.g. low self-efficacy) and preferences to avoid the outdoors (e.g. don't like hot/cold weather, getting dirty, chaos of playground). Because individual teachers determine daily schedules and ultimately make the decision whether to take the children outdoors, they serve as gatekeepers to the playground. Participants discussed a spectrum of roles on the playground, from facilitator to chaperone to physical activity inhibitor. These findings suggest that children could have very different gross motor experiences even within the same facility (with presumably the same environment and policies), based on the beliefs, creativity and level of engagement of their teacher. PMID:21804083

  7. Making Physics Fun: Key Concepts, Classroom Activities, and Everyday Examples, Grades K-8

    ERIC Educational Resources Information Center

    Prigo, Robert

    2007-01-01

    Teaching physical science in the elementary and middle grades can be challenging for busy teachers faced with growing science demands and limited classroom resources. Robert Prigo provides fun and engaging activities using safe, available materials that educators can easily incorporate into lesson plans. Extensive examples, sample inquiry…

  8. Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3.

    PubMed Central

    Rietveld, L E; Holthuizen, P E; Sussenbach, J S

    1997-01-01

    Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concluded that Box A is essential for P3 activity. Box A is part of a region 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functional analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3. PMID:9581544

  9. The time of day differently influences fatigue and locomotor activity: is body temperature a key factor?

    PubMed

    Machado, Frederico Sander Mansur; Rodovalho, Gisele Vieira; Coimbra, Cândido Celso

    2015-03-01

    The aim of this study was to verify the possible interactions between exercise capacity and spontaneous locomotor activity (SLA) during the oscillation of core body temperature (Tb) that occurs during the light/dark cycle. Wistar rats (n=11) were kept at an animal facility under a light/dark cycle of 14/10h at an ambient temperature of 23°C and water and food ad libitum. Initially, in order to characterize the daily oscillation in SLA and Tb of the rats, these parameters were continuously recorded for 24h using an implantable telemetric sensor (G2 E-Mitter). The animals were randomly assigned to two progressive exercise test protocols until fatigue during the beginning of light and dark-phases. Fatigue was defined as the moment rats could not keep pace with the treadmill. We assessed the time to fatigue, workload and Tb changes induced by exercise. Each test was separated by 3days. Our results showed that exercise capacity and heat storage were higher during the light-phase (p<0.05). In contrast, we observed that both SLA and Tb were higher during the dark-phase (p<0.01). Notably, the correlation analysis between the amount of SLA and the running capacity observed at each phase of the daily cycle revealed that, regardless of the time of the day, both types of locomotor physical activity have an important inherent component (r=0.864 and r=0.784, respectively, p<0.01) without a direct relationship between them. This finding provides further support for the existence of specific control mechanisms for each type of physical activity. In conclusion, our data indicate that the relationship between the body temperature and different types of physical activity might be affected by the light/dark cycle. These results mean that, although exercise performance and spontaneous locomotor activity are not directly associated, both are strongly influenced by daily cycles of light and dark. PMID:25479573

  10. Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

    PubMed Central

    Ulianov, Sergey V.; Khrameeva, Ekaterina E.; Gavrilov, Alexey A.; Flyamer, Ilya M.; Kos, Pavel; Mikhaleva, Elena A.; Penin, Aleksey A.; Logacheva, Maria D.; Imakaev, Maxim V.; Chertovich, Alexander; Gelfand, Mikhail S.; Shevelyov, Yuri Y.; Razin, Sergey V.

    2016-01-01

    Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)+ RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin. PMID:26518482

  11. Improving upon Nature: Active site remodeling produces highly efficient aldolase activity towards hydrophobic electrophilic substrates

    PubMed Central

    Cheriyan, Manoj; Toone, Eric J.; Fierke, Carol A.

    2012-01-01

    Substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S/S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (S-KHPB). These mutations improve the value of kcat/KMS-KHPB by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of kcatS-KHPB for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximum value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate (KHO), a non-functionalized hydrophobic analog. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases. Furthermore, targeting mutations to the active site provides marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to naturally occurring enzymes. PMID

  12. Atomically-thin two-dimensional sheets for understanding active sites in catalysis.

    PubMed

    Sun, Yongfu; Gao, Shan; Lei, Fengcai; Xie, Yi

    2015-02-01

    Catalysis can speed up chemical reactions and it usually occurs on the low coordinated steps, edges, terraces, kinks and corner atoms that are often called "active sites". However, the atomic level interplay between active sites and catalytic activity is still an open question, owing to the large difference between idealized models and real catalysts. This stimulates us to pursue a suitable material model for studying the active sites-catalytic activity relationship, in which the atomically-thin two-dimensional sheets could serve as an ideal model, owing to their relatively simple type of active site and the ultrahigh fraction of active sites that are comparable to the overall atoms. In this tutorial review, we focus on the recent progress in disclosing the factors that affect the activity of reactive sites, including characterization of atomic coordination number, structural defects and disorder in ultrathin two-dimensional sheets by X-ray absorption fine structure spectroscopy, positron annihilation spectroscopy, electron spin resonance and high resolution transmission electron microscopy. Also, we overview their applications in CO catalytic oxidation, photocatalytic water splitting, electrocatalytic oxygen and hydrogen evolution reactions, and hence highlight the atomic level interplay among coordination number, structural defects/disorder, active sites and catalytic activity in the two-dimensional sheets with atomic thickness. Finally, we also present the major challenges and opportunities regarding the role of active sites in catalysis. We believe that this review provides critical insights for understanding the catalysis and hence helps to develop new catalysts with high catalytic activity. PMID:25382246

  13. The active sites of supported silver particle catalysts in formaldehyde oxidation.

    PubMed

    Chen, Yaxin; Huang, Zhiwei; Zhou, Meijuan; Hu, Pingping; Du, Chengtian; Kong, Lingdong; Chen, Jianmin; Tang, Xingfu

    2016-08-01

    Surface silver atoms with upshifted d-orbitals are identified as the catalytically active sites in formaldehyde oxidation by correlating their activity with the number of surface silver atoms, and the degree of the d-orbital upshift governs the catalytic performance of the active sites. PMID:27406403

  14. Effect of feed starvation on side-stream anammox activity and key microbial populations.

    PubMed

    Reeve, Petra J; Mouilleron, Irina; Chuang, Hui-Ping; Thwaites, Ben; Hyde, Kylie; Dinesh, Nirmala; Krampe, Joerg; Lin, Tsair-Fuh; van den Akker, Ben

    2016-04-15

    The anaerobic ammonium oxidation (anammox) process is widely acknowledged to be susceptible to a wide range of environmental factors given the slow growth rate of the anammox bacteria. Surprisingly there is limited experimental data regarding the susceptibility of the anammox process to feed starvations which may be encountered in full-scale applications. Therefore, a study was established to investigate the impact of feed starvations on nitritation and anammox activity in a demonstration-scale sequencing batch reactor. Three starvation periods were trialled, lasting one fortnight (15 d), one month (33 d) and two months (62 d). Regardless of the duration of the starvation period, assessment of the ammonia removal performance demonstrated nitritation and anammox activity were reinstated within one day of recovery operation. Characterisation of the community structure using 16S rRNA and functional genes specific for nitrogen-related microbes showed there was no clear impact or shift in the microbial populations between starvation and recovery phases. PMID:26861222

  15. Is Nox4 a key regulator of the activated state of fibroblasts in systemic sclerosis?

    PubMed

    Böhm, Markus; Dosoki, Heba; Kerkhoff, Claus

    2014-09-01

    The family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases consists of phagocytic gp91(phox) and six-related isoforms. Recent evidence indicates that the NADPH oxidase isoform Nox4 controls vascular, renal and pulmonary injury. We propose that Nox4 is an intrinsic regulator of the activated state of dermal fibroblasts in systemic sclerosis (SSc). Profibrotic cytokines on the one hand and antifibrogenic factors such as α-melanocyte-stimulating hormone on the other hand may target Nox4 as an intracellular nodal point. Via increased or decreased generation of reactive oxygen species and/or hydrogen peroxide, Nox4 could orchestrate collagen synthesis, differentiation of dermal fibroblasts into a profibrotic myofibroblast phenotype and thus dermal fibrosis. Confirmation of this hypothesis will have important consequences in our understanding of the activated state of dermal fibroblasts in SSc. Based on the availability of clinically useful Nox4 inhibitors, novel antifibrotic therapies of SSc can be envisioned. PMID:25040787

  16. 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

  17. Fluid Geochemistry of the Capelinhos Vent Site. A Key to Understand the Lucky Strike Hydrothermal Vent Field (37°N, MAR).

    NASA Astrophysics Data System (ADS)

    Leleu, T.; Chavagnac, V.; Cannat, M.; Ceuleneer, G.; Castillo, A.; Menjot, L.

    2015-12-01

    The Lucky Strike hydrothermal field is situated at the mid-Atlantic ridge, south of the Azores, on top of a central volcano within the axial valley. The volcano is composed of a fossil lava lake surrounded by three volcanic cones. An Axial Magma Chamber (AMC) is reported 3.4km below the seafloor. The active venting sites are situated around the fossil lava lake and are directly linked to the heat supplied by the AMC. High temperature fluids from the Lucky Strike field were sampled in 2013, 2014 and 2015 in order to document the depth of the reaction zone, subsurface mixing, geographical control and magmatic degassing. A new active site named Capelinhos was discovered approximately 1.5km eastward from the lava lake, during exploration by ROV Victor6000 - MoMARsat cruise, 2013. It is composed of 10m-high chimneys discharging black smoker-type fluid. Fluid temperatures were 328°C in 2013 and decreased to 318°C in 2014 and 2015. Capelinhos fluids are Cl-depleted by 55% compared to seawater indicating phase separation at depth. In comparison, the other sites range from 6% enrichment (2608/Y3 site) to 22% depletion (Eiffel tower site). Si geothermobarometry of Y3 site estimates quartz equilibration at P=300 bars and T=360-380°C, coherent with Fe/Mn geothermometer (T=370±10°C). For Capelinhos, Fe/Mn suggests 398°C (±10°C) which is close to the critical point of seawater (P=300 bars and T=407°C). Other geothermobarometer uses Si/Cl vapor-like fluid to constrain depth of the top of reaction zone and predicts significant bias due to mixing along the up-flow zone. Application gives P=~370 bars, T=~435°C at Capelinhos and P=~390 bars, T=~440°C at Eiffel tower. This is further sustained by end-member 87Sr/86Sr=0.7038, which indicates little interaction of Capelinhos vent fluids with seawater-derived fluid, compared to other vapor-like sites with 87Sr/86Sr=0.7043. Because of its external location, Capelinhos site isn't influenced by the complex tectonic context of the

  18. In situ probing of the active site geometry of ultrathin nanowires for the oxygen reduction reaction

    DOE PAGESBeta

    Liu, Haiqing; Wong, Stanislaus S.; An, Wei; Li, Yuanyuan; Frenkel, Anatoly I.; Sasaki, Kotaro; Koenigsmann, Christopher; Su, Dong; Anderson, Rachel M.; Crooks, Richard M.; et al

    2015-09-24

    To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (~2 nm) core–shell Pt~Pd9Au nanowires, which have been previously shownmore » to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu~Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Thus, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general.« less

  19. In situ probing of the active site geometry of ultrathin nanowires for the oxygen reduction reaction

    SciTech Connect

    Liu, Haiqing; Wong, Stanislaus S.; An, Wei; Li, Yuanyuan; Frenkel, Anatoly I.; Sasaki, Kotaro; Koenigsmann, Christopher; Su, Dong; Anderson, Rachel M.; Crooks, Richard M.; Adzic, Radoslav R.; Liu, Ping

    2015-09-24

    To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (~2 nm) core–shell Pt~Pd9Au nanowires, which have been previously shown to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu~Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Thus, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general.

  20. In Situ Probing of the Active Site Geometry of Ultrathin Nanowires for the Oxygen Reduction Reaction.

    PubMed

    Liu, Haiqing; An, Wei; Li, Yuanyuan; Frenkel, Anatoly I; Sasaki, Kotaro; Koenigsmann, Christopher; Su, Dong; Anderson, Rachel M; Crooks, Richard M; Adzic, Radoslav R; Liu, Ping; Wong, Stanislaus S

    2015-10-01

    To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (∼2 nm) core-shell Pt∼Pd9Au nanowires, which have been previously shown to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu∼Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Hence, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general. PMID:26402364

  1. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  2. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    SciTech Connect

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  3. Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity[W

    PubMed Central

    Shumskaya, Maria; Bradbury, Louis M.T.; Monaco, Regina R.; Wurtzel, Eleanore T.

    2012-01-01

    Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zea mays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis. PMID:23023170

  4. Cell proliferation is a key determinant of the outcome of FOXO3a activation

    SciTech Connect

    Poulsen, Raewyn C. Carr, Andrew J.; Hulley, Philippa A.

    2015-06-19

    The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media, FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail. - Highlights: • FOXO3a activity can result in either promotion of cell survival or apoptosis. • The outcome of FOXO3a activation differs in proliferating compared to non-proliferating cells. • Proliferating

  5. Identification of Key Residues for pH Dependent Activation of Violaxanthin De-Epoxidase from Arabidopsis thaliana

    PubMed Central

    Fufezan, Christian; Simionato, Diana; Morosinotto, Tomas

    2012-01-01

    Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pKa values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121–Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role. PMID:22558195

  6. Estrogen-related receptor gamma is a key regulator of muscle mitochondrial activity and oxidative capacity.

    PubMed

    Rangwala, Shamina M; Wang, Xiaomei; Calvo, Jennifer A; Lindsley, Loren; Zhang, Yunyu; Deyneko, Galina; Beaulieu, Valerie; Gao, Jiaping; Turner, Gordon; Markovits, Judit

    2010-07-16

    Estrogen-related receptor gamma (ERRgamma) regulates the perinatal switch to oxidative metabolism in the myocardium. We wanted to understand the significance of induction of ERRgamma expression in skeletal muscle by exercise. Muscle-specific VP16ERRgamma transgenic mice demonstrated an increase in exercise capacity, mitochondrial enzyme activity, and enlarged mitochondria despite lower muscle weights. Furthermore, peak oxidative capacity was higher in the transgenics as compared with control littermates. In contrast, mice lacking one copy of ERRgamma exhibited decreased exercise capacity and muscle mitochondrial function. Interestingly, we observed that increased ERRgamma in muscle generates a gene expression profile that closely overlays that of red oxidative fiber-type muscle. We further demonstrated that a small molecule agonist of ERRbeta/gamma can increase mitochondrial function in mouse myotubes. Our data indicate that ERRgamma plays an important role in causing a shift toward slow twitch muscle type and, concomitantly, a greater capacity for endurance exercise. Thus, the activation of this nuclear receptor provides a potential node for therapeutic intervention for diseases such as obesity, which is associated with reduced oxidative metabolism and a lower type I fiber content in skeletal muscle. PMID:20418374

  7. Estrogen-related Receptor γ Is a Key Regulator of Muscle Mitochondrial Activity and Oxidative Capacity

    PubMed Central

    Rangwala, Shamina M.; Wang, Xiaomei; Calvo, Jennifer A.; Lindsley, Loren; Zhang, Yunyu; Deyneko, Galina; Beaulieu, Valerie; Gao, Jiaping; Turner, Gordon; Markovits, Judit

    2010-01-01

    Estrogen-related receptor γ (ERRγ) regulates the perinatal switch to oxidative metabolism in the myocardium. We wanted to understand the significance of induction of ERRγ expression in skeletal muscle by exercise. Muscle-specific VP16ERRγ transgenic mice demonstrated an increase in exercise capacity, mitochondrial enzyme activity, and enlarged mitochondria despite lower muscle weights. Furthermore, peak oxidative capacity was higher in the transgenics as compared with control littermates. In contrast, mice lacking one copy of ERRγ exhibited decreased exercise capacity and muscle mitochondrial function. Interestingly, we observed that increased ERRγ in muscle generates a gene expression profile that closely overlays that of red oxidative fiber-type muscle. We further demonstrated that a small molecule agonist of ERRβ/γ can increase mitochondrial function in mouse myotubes. Our data indicate that ERRγ plays an important role in causing a shift toward slow twitch muscle type and, concomitantly, a greater capacity for endurance exercise. Thus, the activation of this nuclear receptor provides a potential node for therapeutic intervention for diseases such as obesity, which is associated with reduced oxidative metabolism and a lower type I fiber content in skeletal muscle. PMID:20418374

  8. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis

    PubMed Central

    Kardon, Julia R.; Yien, Yvette Y.; Huston, Nicholas C.; Branco, Diana S.; Hildick-Smith, Gordon J.; Rhee, Kyu Y.; Paw, Barry H.; Baker, Tania A.

    2015-01-01

    SUMMARY The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is five-fold reduced in yeast lacking mtClpX activity, and total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX therefore is a widely conserved stimulator of an essential biosynthetic pathway, and employs a previously unrecognized mechanism for AAA+ unfoldases. PMID:25957689

  9. Activities of Daily Living in Mexican American Caregivers: The Key to Continuing Informal Care

    PubMed Central

    Evans, Bronwynne C.; Belyea, Michael J.; Coon, David W.; Ume, Ebere

    2013-01-01

    La familia drives elder care in Mexican–American (MA) families, but nursing home placement can result from day-to-day caregiving demands that increase caregiver difficulty with activities of daily living (ADLs). Using life course perspective, this article describes the initial data wave of 31 MA caregivers from a descriptive, longitudinal, mixed-methods study of 110 MA caregivers and care recipients over 15 months in their caregiving trajectories. Fifteen of 31 caregivers consistently indicated “no help needed” on the Katz ADL, whereas all but one reported “help needed” during semistructured interviews with cultural brokers. In addition to the discrepancy between results on the Katz ADL and interviews, findings include consideration of nursing home placement by moderately acculturated caregivers and minimization of their illnesses by caregivers. Additional methods of MA caregiver assessment may be needed due to the questionable accuracy of the Katz ADL; additional research should explore minimization and acculturation in MA caregivers. PMID:22740307

  10. The flavonoid paradox: conjugation and deconjugation as key steps for the biological activity of flavonoids.

    PubMed

    Perez-Vizcaino, Francisco; Duarte, Juan; Santos-Buelga, Celestino

    2012-07-01

    Flavonoids have been proposed to exert beneficial effects in the prevention of a large number of diseases, including cancer, cardiovascular disease, and neurodegenerative disorders. Paradoxically, despite the most representative flavonoid--quercetin--exerting biologically demonstrable systemic effects, it is not found in plasma after oral administration and its circulating metabolites show weak activity in vitro. The current available evidence indicates that quercetin is extensively metabolized into methylated and glucurono- and sulfo-conjugated metabolites, which are the plasma circulating forms; and glucurono-, but not sulfo-conjugates, can be hydrolyzed at the vascular level, yielding the parent aglycone which accumulates in tissues. Thus conjugation is a reversible process and, at least regarding the vasodilator and antihypertensive effects, the conjugation-deconjugation cycle appears to be an absolute requirement. Glucuronidated derivatives transport quercetin and its methylated form, and deliver to the tissues the free aglycone, which is the final effector. PMID:22555950

  11. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  12. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... from leasing, site characterization, and site assessment in and around the Call Area (76 FR 51391). The... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  13. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore..., site characterization, and site assessment in and around the Call Area (76 FR 51391). The Call Area is... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  14. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  15. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

    This paper, presented as a poster, identifies why ballistic protection should now be considered at nuclear sites to counter terrorist threats. A proven and flexible form of multi purpose protection is described in detail with identification of trial results that show its suitability for this role. (authors)

  16. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    SciTech Connect

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  17. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  18. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  19. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

  20. A Detailed Data-Driven Network Model of Prefrontal Cortex Reproduces Key Features of In Vivo Activity.

    PubMed

    Hass, Joachim; Hertäg, Loreen; Durstewitz, Daniel

    2016-05-01

    The prefrontal cortex is centrally involved in a wide range of cognitive functions and their impairment in psychiatric disorders. Yet, the computational principles that govern the dynamics of prefrontal neural networks, and link their physiological, biochemical and anatomical properties to cognitive functions, are not well understood. Computational models can help to bridge the gap between these different levels of description, provided they are sufficiently constrained by experimental data and capable of predicting key properties of the intact cortex. Here, we present a detailed network model of the prefrontal cortex, based on a simple computationally efficient single neuron model (simpAdEx), with all parameters derived from in vitro electrophysiological and anatomical data. Without additional tuning, this model could be shown to quantitatively reproduce a wide range of measures from in vivo electrophysiological recordings, to a degree where simulated and experimentally observed activities were statistically indistinguishable. These measures include spike train statistics, membrane potential fluctuations, local field potentials, and the transmission of transient stimulus information across layers. We further demonstrate that model predictions are robust against moderate changes in key parameters, and that synaptic heterogeneity is a crucial ingredient to the quantitative reproduction of in vivo-like electrophysiological behavior. Thus, we have produced a physiologically highly valid, in a quantitative sense, yet computationally efficient PFC network model, which helped to identify key properties underlying spike time dynamics as observed in vivo, and can be harvested for in-depth investigation of the links between physiology and cognition. PMID:27203563

  1. A Detailed Data-Driven Network Model of Prefrontal Cortex Reproduces Key Features of In Vivo Activity

    PubMed Central

    Hass, Joachim; Hertäg, Loreen; Durstewitz, Daniel

    2016-01-01

    The prefrontal cortex is centrally involved in a wide range of cognitive functions and their impairment in psychiatric disorders. Yet, the computational principles that govern the dynamics of prefrontal neural networks, and link their physiological, biochemical and anatomical properties to cognitive functions, are not well understood. Computational models can help to bridge the gap between these different levels of description, provided they are sufficiently constrained by experimental data and capable of predicting key properties of the intact cortex. Here, we present a detailed network model of the prefrontal cortex, based on a simple computationally efficient single neuron model (simpAdEx), with all parameters derived from in vitro electrophysiological and anatomical data. Without additional tuning, this model could be shown to quantitatively reproduce a wide range of measures from in vivo electrophysiological recordings, to a degree where simulated and experimentally observed activities were statistically indistinguishable. These measures include spike train statistics, membrane potential fluctuations, local field potentials, and the transmission of transient stimulus information across layers. We further demonstrate that model predictions are robust against moderate changes in key parameters, and that synaptic heterogeneity is a crucial ingredient to the quantitative reproduction of in vivo-like electrophysiological behavior. Thus, we have produced a physiologically highly valid, in a quantitative sense, yet computationally efficient PFC network model, which helped to identify key properties underlying spike time dynamics as observed in vivo, and can be harvested for in-depth investigation of the links between physiology and cognition. PMID:27203563

  2. Strong Nonadditivity as a Key Structure–Activity Relationship Feature: Distinguishing Structural Changes from Assay Artifacts

    PubMed Central

    2015-01-01

    Nonadditivity in protein–ligand affinity data represents highly instructive structure–activity relationship (SAR) features that indicate structural changes and have the potential to guide rational drug design. At the same time, nonadditivity is a challenge for both basic SAR analysis as well as many ligand-based data analysis techniques such as Free-Wilson Analysis and Matched Molecular Pair analysis, since linear substituent contribution models inherently assume additivity and thus do not work in such cases. While structural causes for nonadditivity have been analyzed anecdotally, no systematic approaches to interpret and use nonadditivity prospectively have been developed yet. In this contribution, we lay the statistical framework for systematic analysis of nonadditivity in a SAR series. First, we develop a general metric to quantify nonadditivity. Then, we demonstrate the non-negligible impact of experimental uncertainty that creates apparent nonadditivity, and we introduce techniques to handle experimental uncertainty. Finally, we analyze public SAR data sets for strong nonadditivity and use recourse to the original publications and available X-ray structures to find structural explanations for the nonadditivity observed. We find that all cases of strong nonadditivity (ΔΔpKi and ΔΔpIC50 > 2.0 log units) with sufficient structural information to generate reasonable hypothesis involve changes in binding mode. With the appropriate statistical basis, nonadditivity analysis offers a variety of new attempts for various areas in computer-aided drug design, including the validation of scoring functions and free energy perturbation approaches, binding pocket classification, and novel features in SAR analysis tools. PMID:25760829

  3. Active site conformational dynamics are coupled to catalysis in the mRNA decapping enzyme Dcp2

    PubMed Central

    Aglietti, Robin A.; Floor, Stephen N.; McClendon, Chris L.; Jacobson, Matthew P.; Gross, John D.

    2013-01-01

    Summary Removal of the 5′ cap structure by Dcp2 is a major step in several 5′–3′ mRNA decay pathways. The activity of Dcp2 is enhanced by Dcp1 and bound coactivators, yet the details of how these interactions are linked to chemistry are poorly understood. Here we report three crystal structures of the catalytic Nudix hydrolase domain of Dcp2 that demonstrate binding of a catalytically essential metal ion, and enzyme kinetics are used to identify several key active site residues involved in acid/base chemistry of decapping. Using NMR and molecular dynamics, we find that a conserved metal binding loop on the catalytic domain undergoes conformational changes during the catalytic cycle. These findings describe key events during the chemical step of decapping, suggest local active site conformational changes are important for activity, and provide a framework to explain stimulation of catalysis by the regulatory domain of Dcp2 and associated coactivators. PMID:23911090

  4. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  5. Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

    PubMed Central

    Grahl, Nora; Demers, Elora G.; Lindsay, Allia K.; Harty, Colleen E.; Willger, Sven D.; Piispanen, Amy E.; Hogan, Deborah A.

    2015-01-01

    Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate

  6. Where's the Noise? Key Features of Spontaneous Activity and Neural Variability Arise through Learning in a Deterministic Network.

    PubMed

    Hartmann, Christoph; Lazar, Andreea; Nessler, Bernhard; Triesch, Jochen

    2015-12-01

    Even in the absence of sensory stimulation the brain is spontaneously active. This background "noise" seems to be the dominant cause of the notoriously high trial-to-trial variability of neural recordings. Recent experimental observations have extended our knowledge of trial-to-trial variability and spontaneous activity in several directions: 1. Trial-to-trial variability systematically decreases following the onset of a sensory stimulus or the start of a motor act. 2. Spontaneous activity states in sensory cortex outline the region of evoked sensory responses. 3. Across development, spontaneous activity aligns itself with typical evoked activity patterns. 4. The spontaneous brain activity prior to the presentation of an ambiguous stimulus predicts how the stimulus will be interpreted. At present it is unclear how these observations relate to each other and how they arise in cortical circuits. Here we demonstrate that all of these phenomena can be accounted for by a deterministic self-organizing recurrent neural network model (SORN), which learns a predictive model of its sensory environment. The SORN comprises recurrently coupled populations of excitatory and inhibitory threshold units and learns via a combination of spike-timing dependent plasticity (STDP) and homeostatic plasticity mechanisms. Similar to balanced network architectures, units in the network show irregular activity and variable responses to inputs. Additionally, however, the SORN exhibits sequence learning abilities matching recent findings from visual cortex and the network's spontaneous activity reproduces the experimental findings mentioned above. Intriguingly, the network's behaviour is reminiscent of sampling-based probabilistic inference, suggesting that correlates of sampling-based inference can develop from the interaction of STDP and homeostasis in deterministic networks. We conclude that key observations on spontaneous brain activity and the variability of neural responses can be

  7. Where’s the Noise? Key Features of Spontaneous Activity and Neural Variability Arise through Learning in a Deterministic Network

    PubMed Central

    Hartmann, Christoph; Lazar, Andreea; Nessler, Bernhard; Triesch, Jochen

    2015-01-01

    Even in the absence of sensory stimulation the brain is spontaneously active. This background “noise” seems to be the dominant cause of the notoriously high trial-to-trial variability of neural recordings. Recent experimental observations have extended our knowledge of trial-to-trial variability and spontaneous activity in several directions: 1. Trial-to-trial variability systematically decreases following the onset of a sensory stimulus or the start of a motor act. 2. Spontaneous activity states in sensory cortex outline the region of evoked sensory responses. 3. Across development, spontaneous activity aligns itself with typical evoked activity patterns. 4. The spontaneous brain activity prior to the presentation of an ambiguous stimulus predicts how the stimulus will be interpreted. At present it is unclear how these observations relate to each other and how they arise in cortical circuits. Here we demonstrate that all of these phenomena can be accounted for by a deterministic self-organizing recurrent neural network model (SORN), which learns a predictive model of its sensory environment. The SORN comprises recurrently coupled populations of excitatory and inhibitory threshold units and learns via a combination of spike-timing dependent plasticity (STDP) and homeostatic plasticity mechanisms. Similar to balanced network architectures, units in the network show irregular activity and variable responses to inputs. Additionally, however, the SORN exhibits sequence learning abilities matching recent findings from visual cortex and the network’s spontaneous activity reproduces the experimental findings mentioned above. Intriguingly, the network’s behaviour is reminiscent of sampling-based probabilistic inference, suggesting that correlates of sampling-based inference can develop from the interaction of STDP and homeostasis in deterministic networks. We conclude that key observations on spontaneous brain activity and the variability of neural responses can be

  8. Sp1 Sites in the Noncoding Control Region of BK Polyomavirus Are Key Regulators of Bidirectional Viral Early and Late Gene Expression

    PubMed Central

    Bethge, Tobias; Hachemi, Helen A.; Manzetti, Julia; Gosert, Rainer; Schaffner, Walter

    2015-01-01

    ABSTRACT In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 28 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bidirectional reporter constructs. Compared to the archetype, group 1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group 2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. Group 3 mutations decreased early and late gene expression and included two other Sp1 sites near the early TSS. Recombinant viruses bearing group 1 NCCRs showed increased replication in human renal epithelial cells similar to clinical rr-NCCR variants. Group 2 and 3 viruses showed intermediate or no replication, respectively. A literature search revealed unnoticed group 1 mutations in BKPyV nephropathy, hemorrhagic cystitis, and disseminated disease. IMPORTANCE The NCCRs of polyomaviruses mediate silent persistence of the viral genome as well as the appropriately timed (re)activation of the viral life cycle. This study indicates that the basal BKPyV NCCR is critically controlled by a hierarchy of single TFBS in the archetype NCCR that direct, modulate, and execute the bidirectional early and late viral gene expression. The results provide new insights into how BKPyV NCCR functions as a viral sensor of host cell signals and shed new light on how transcription factors like Sp1 control bidirectional viral gene expression and contribute to replication and pathology

  9. Disk-driven hydromagnetic winds as a key ingredient of active galactic nuclei unification schemes

    NASA Technical Reports Server (NTRS)

    Konigl, Arieh; Kartje, John F.

    1994-01-01

    Centrifugally driven winds from the surfaces of magnetized accretion disks have been recognized as an attractive mechanism of removing the angular momentum of the accreted matter and of producing the bipolar outflows and jets that are often associated with compact astronomical objects. As previously suggested in the context of young stellar objects, such winds have unique observational manifestations stemming from their highly stratified density and velocity structure and from their exposure to the strong continuum radiation field of the compact object. We have applied this scenario to active galactic nuclei (AGNs) and investigated the properties of hydromagnetic outflows that originate within approximately 10(M(sub 8)) pc of the central 10(exp 8)(M(sub 8)) solar mass black hole. On the basis of our results, we propose that hydromagnetic disk-driven winds may underlie the classification of broad-line and narrow-line AGNs (e.g., the Seyfert 1/Seyfert 2 dichotomy) as well as the apparent dearth of luminous Seyfert 2 galaxies. More generally, we demonstrate that such winds could strongly influence the spectral characteristics of Seyfert galaxies, QSOs, and BL Lac objects (BLOs). In our picture, the torus is identified with the outer regions of the wind where dust uplifted from the disk surfaces by gas-grain collisions is embedded in the outflow. Using an efficient radiative transfer code, we show that the infrared emission of Seyfert galaxies and QSOs can be attributed to the reprocessing of the UV/soft X-ray AGN continuum by the dust in the wind and the disk. We demonstrate that the radiation pressure force flattens the dust distribution in objects with comparatively high (but possibly sub-Eddington) bolometric luminosities, and we propose this as one likely reason for the apparent paucity of narrow-line objects among certain high-luminosity AGNs. Using the XSTAR photoionization code, we show that the inner regions of the wind could naturally account for the warm

  10. Surface Snowpack Key to Bromine Activation in a Changing Arctic Environment

    NASA Astrophysics Data System (ADS)

    Pratt, Kerri; Custard, Kyle; Shepson, Paul; Douglas, Thomas; Pöhler, Denis; Stephan, General; Zielcke, Johannes; Simpson, William; Platt, Ulrich; von Glasow, Roland; Tanner, David; Huey, L. Gregory; Carlsen, Mark; Stirm, Brian

    2013-04-01

    Arctic sea ice is rapidly declining and transforming from a multiyear ice pack to thinner, more saline, seasonal ice, which has important implications for Arctic atmospheric composition. Following springtime polar sunrise, "ozone depletion events", attributed to bromine chemistry, lead to episodic decreases in lower tropospheric ozone concentrations to near zero, concurrent with mercury depletion and deposition. Despite our increasing understanding of the spatial variability of BrO and possible reaction pathways based on laboratory studies, important questions remain regarding the most efficient sources of and mechanisms for Arctic halogen activation. During the March-April 2012 BRomine, Ozone, and Mercury EXperiment (BROMEX) in Barrow, Alaska, outdoor chamber experiments with snow and ice samples were conducted. Ozone was added as the precursor oxidant, and the samples were investigated with and without ambient sunlight. Samples included first-year sea ice, brine icicles, several layers of snow above first-year sea ice, and seasonal snow above the tundra. Chemical ionization mass spectrometry was utilized to monitor Br2 production. Tundra snow and surface snow above sea ice produced the most Br2, with no production resulting from sea ice and basal snow directly above sea ice. Overall, the most efficient Br2 production was observed from snow samples characterized by lower pH and higher bromide/chloride ratios. Br2 was only observed in the presence of sunlight, indicating the role of snowpack photochemical reactions and the hydroxyl radical in its production. Br2 production via the surface snowpack explains previously-observed BrO enhancements above sea ice, as well as observations of inland tundra hotspots in measured BrO by aircraft-based nadir MAX-DOAS (Multi Axis-Differential Optical Absorption Spectroscopy) measurements, conducted during BROMEX. The findings indicate that atmospherically processed snow is likely a major source of Arctic bromine release, which

  11. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  12. Avoidance and activation as keys to depression: adaptation of the Behavioral Activation for Depression Scale in a Spanish sample.

    PubMed

    Barraca, Jorge; Pérez-Alvarez, Marino; Lozano Bleda, José Héctor

    2011-11-01

    In this paper we present the adaptation of the Behavioral Activation for Depression Scale (BADS), developed by Kanter, Mulick, Busch, Berlin, and Martell (2007), in a Spanish sample. The psychometric properties were tested in a sample of 263 participants (124 clinical and 139 non-clinical). The results show that, just as in the original English version, the Spanish BADS is a valid and internally consistent scale. Construct validity was examined by correlation with the BDI-II, AAQ, ATQ, MCQ-30, STAI and EROS. Factor analysis justified the four-dimensions of the original instrument (Activation, Avoidance/Rumination, Work/School Impairment and Social Impairment), although with some differences in the factor loadings of the items. Further considerations about the usefulness of the BADS in the clinical treatment of depressed patients are also suggested. PMID:22059343

  13. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. PMID:26620444

  14. Active sites and mechanisms for H2O2 decomposition over Pd catalysts.

    PubMed

    Plauck, Anthony; Stangland, Eric E; Dumesic, James A; Mavrikakis, Manos

    2016-04-01

    A combination of periodic, self-consistent density functional theory (DFT-GGA-PW91) calculations, reaction kinetics experiments on a SiO2-supported Pd catalyst, and mean-field microkinetic modeling are used to probe key aspects of H2O2 decomposition on Pd in the absence of cofeeding H2 We conclude that both Pd(111) and OH-partially covered Pd(100) surfaces represent the nature of the active site for H2O2 decomposition on the supported Pd catalyst reasonably well. Furthermore, all reaction flux in the closed catalytic cycle is predicted to flow through an O-O bond scission step in either H2O2 or OOH, followed by rapid H-transfer steps to produce the H2O and O2 products. The barrier for O-O bond scission is sensitive to Pd surface structure and is concluded to be the central parameter governing H2O2 decomposition activity. PMID:27006504

  15. Current Challenges for Modeling Enzyme Active Sites by Biomimetic Synthetic Diiron Complexes

    PubMed Central

    Friedle, Simone; Reisner, Erwin; Lippard, Stephen J.

    2010-01-01

    This tutorial review describes recent progress in modeling the active sites of carboxylate-rich non-heme diiron enzymes that activate dioxygen to carry out several key reactions in nature. The chemistry of soluble methane monooxygenase, which catalyzes the selective oxidation of methane to methanol, is of particular interest for (bio)technological applications. Novel synthetic diiron complexes that mimic structural, and, to a lesser extent, functional features of these diiron enzymes are discussed. The chemistry of the enzymes is also briefly summarized. A particular focus of this review is on models that mimic characteristics of the diiron systems that were previously not emphasized, including systems that contain (i) aqua ligands, (ii) different substrates tethered to the ligand framework, (iii) dendrimers attached to carboxylates to mimic the protein environment, (iv) two N-donors in a syn-orientation with respect to the iron-iron vector, and (v) a N-rich ligand environment capable of accessing oxygenated high-valent diiron intermediates. PMID:20485834

  16. Synthetic Active Site Model of the [NiFeSe] Hydrogenase

    PubMed Central

    Wombwell, Claire; Reisner, Erwin

    2015-01-01

    A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe(‘S2Se2’)(CO)3] (H2‘S2Se2’=1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni(‘S2Se2’)] with [Fe(CO)3bda] (bda=benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe(‘S2Se2’)(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe(‘S2Se2’)(CO)3] with the previously reported thiolate analogue [NiFe(‘S4’)(CO)3] (H2‘S4’=H2xbsms=1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe(‘S2Se2’)(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe(‘S2Se2’)(CO)3] and [NiFe(‘S4’)(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. PMID:25847470

  17. Revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison

    PubMed Central

    Chen, Luonan; Wu, Ling-Yun; Wang, Yong; Zhang, Shihua; Zhang, Xiang-Sun

    2006-01-01

    Background Protein structure comparison is one of the most important problems in computational biology and plays a key role in protein structure prediction, fold family classification, motif finding, phylogenetic tree reconstruction and protein docking. Results We propose a novel method to compare the protein structures in an accurate and efficient manner. Such a method can be used to not only reveal divergent evolution, but also identify circular permutations and further detect active-sites. Specifically, we define the structure alignment as a multi-objective optimization problem, i.e., maximizing the number of aligned atoms and minimizing their root mean square distance. By controlling a single distance-related parameter, theoretically we can obtain a variety of optimal alignments corresponding to different optimal matching patterns, i.e., from a large matching portion to a small matching portion. The number of variables in our algorithm increases with the number of atoms of protein pairs in almost a linear manner. In addition to solid theoretical background, numerical experiments demonstrated significant improvement of our approach over the existing methods in terms of quality and efficiency. In particular, we show that divergent evolution, circular permutations and active-sites (or structural motifs) can be identified by our method. The software SAMO is available upon request from the authors, or from and . Conclusion A novel formulation is proposed to accurately align protein structures in the framework of multi-objective optimization, based on a sequence order-independent strategy. A fast and accurate algorithm based on the bipartite matching algorithm is developed by exploiting the special features. Convergence of computation is shown in experiments and is also theoretically proven. PMID:16948858

  18. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  19. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  20. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

  1. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  2. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

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

    NASA Astrophysics Data System (ADS)

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

    2006-06-01

    Vertical distributions of the potential activities of some key enzymes mediating nitrification and denitrification were investigated within the oxygen (O 2) minimum zone of the Arabian Sea at a number of locations between latitudes 17°N and 21°N and longitudes 63°E and 68°E so as to get an insight into the predominant biochemical mode(s) of production and consumption of nitrous oxide (N 2O). Results revealed that the dissimilatory nitrate (NO -3) reduction activity was generally very low or absent within the σ θ range 26.6-26.8, which corresponds to the Persian Gulf Watermass (PGW). Depth profiles of nitrate reductase (NaR), nitrite reductase (NiR) and ammonia monooxygenase (AMO) activities were compared with those of O 2, NO -3, nitrite (NO -2) and N 2O, and it is concluded that nitrifier denitrification rather than heterotrophic denitrification is active within the core of PGW. The presence of multiple peaks of AMO activity coinciding with distinct maxima in the O 2 profile and with a trend opposite to that of NaR activity indicates that the two processes, viz., classical and nitrifier denitrification, occur in discrete layers, probably determined by the variations in the ambient O 2 concentrations at various depths surrounding the PGW core. Further, it appears that at the depths where nitrifier denitrification is active in the absence of heterotrophic denitrification, N 2O builds up as its consumption may be inhibited by O 2. Possible reasons for the occurrence of appreciable nitrate deficit within the core of PGW, where dissimilatory NO -3 reduction is lacking, are discussed.

  4. The importance of delineating networks by activity type in bottlenose dolphins (Tursiops truncatus) in Cedar Key, Florida

    PubMed Central

    Gazda, Stefanie; Iyer, Swami; Killingback, Timothy; Connor, Richard; Brault, Solange

    2015-01-01

    Network analysis has proved to be a valuable tool for studying the behavioural patterns of complex social animals. Often such studies either do not distinguish between different behavioural states of the organisms or simply focus attention on a single behavioural state to the exclusion of all others. In either of these approaches it is impossible to ascertain how the behavioural patterns of individuals depend on the type of activity they are engaged in. Here we report on a network-based analysis of the behavioural associations in a population of bottlenose dolphins (Tursiops truncatus) in Cedar Key, Florida. We consider three distinct behavioural states—socializing, travelling and foraging—and analyse the association networks corresponding to each activity. Moreover, in constructing the different activity networks we do not simply record a spatial association between two individuals as being either present or absent, but rather quantify the degree of any association, thus allowing us to construct weighted networks describing each activity. The results of these weighted activity networks indicate that networks can reveal detailed patterns of bottlenose dolphins at the population level; dolphins socialize in large groups with preferential associations; travel in small groups with preferential associates; and spread out to forage in very small, weakly connected groups. There is some overlap in the socialize and travel networks but little overlap between the forage and other networks. This indicates that the social bonds maintained in other activities are less important as they forage on dispersed, solitary prey. The overall network, not sorted by activity, does not accurately represent any of these patterns. PMID:26064611

  5. The importance of delineating networks by activity type in bottlenose dolphins (Tursiops truncatus) in Cedar Key, Florida.

    PubMed

    Gazda, Stefanie; Iyer, Swami; Killingback, Timothy; Connor, Richard; Brault, Solange

    2015-03-01

    Network analysis has proved to be a valuable tool for studying the behavioural patterns of complex social animals. Often such studies either do not distinguish between different behavioural states of the organisms or simply focus attention on a single behavioural state to the exclusion of all others. In either of these approaches it is impossible to ascertain how the behavioural patterns of individuals depend on the type of activity they are engaged in. Here we report on a network-based analysis of the behavioural associations in a population of bottlenose dolphins (Tursiops truncatus) in Cedar Key, Florida. We consider three distinct behavioural states-socializing, travelling and foraging-and analyse the association networks corresponding to each activity. Moreover, in constructing the different activity networks we do not simply record a spatial association between two individuals as being either present or absent, but rather quantify the degree of any association, thus allowing us to construct weighted networks describing each activity. The results of these weighted activity networks indicate that networks can reveal detailed patterns of bottlenose dolphins at the population level; dolphins socialize in large groups with preferential associations; travel in small groups with preferential associates; and spread out to forage in very small, weakly connected groups. There is some overlap in the socialize and travel networks but little overlap between the forage and other networks. This indicates that the social bonds maintained in other activities are less important as they forage on dispersed, solitary prey. The overall network, not sorted by activity, does not accurately represent any of these patterns. PMID:26064611

  6. Observing large-scale solar surface flows with GONG: Investigation of a key element in solar activity buildup

    NASA Technical Reports Server (NTRS)

    Beck, John G.; Simon, George W.; Hathaway, David H.

    1996-01-01

    The Global Oscillation Network Group (GONG) solar telescope network has begun regular operations, and will provide continuous Doppler images of large-scale nearly-steady motions at the solar surface, primarily those due to supergranulation. Not only the Sun's well-known magnetic network, but also flux diffusion, dispersal, and concentration at the surface appear to be controlled by supergranulation. Through such magnetoconvective interactions, magnetic stresses develop, leading to solar activity. We show a Doppler movie made from a 45.5 hr time series obtained 1995 May 9-10 using data from three of the six GONG sites (Learmonth, Tenerife, Tucson), to demonstrate the capability of this system.

  7. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts.

    PubMed

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and (57)Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  8. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    NASA Astrophysics Data System (ADS)

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-10-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity.

  9. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  10. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. PMID:26786892

  11. Assessment of activation products in the Savannah River Site environment

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

  12. Perinucleolar relocalization and nucleolin as crucial events in the transcriptional activation of key genes in mantle cell lymphoma.

    PubMed

    Allinne, Jeanne; Pichugin, Andrei; Iarovaia, Olga; Klibi, Manel; Barat, Ana; Zlotek-Zlotkiewicz, Ewa; Markozashvili, Diana; Petrova, Natalia; Camara-Clayette, Valérie; Ioudinkova, Elena; Wiels, Joëlle; Razin, Sergey V; Ribrag, Vincent; Lipinski, Marc; Vassetzky, Yegor S

    2014-03-27

    In mantle cell lymphoma (MCL), one allele of the cyclin D1 (Ccnd1) gene is translocated from its normal localization on chromosome 11 to chromosome 14. This is considered as the crucial event in the transformation process of a normal naive B-cell; however, the actual molecular mechanism leading to Ccnd1 activation remains to be deciphered. Using a combination of three-dimensional and immuno-fluorescence in situ hybridization experiments, the radial position of the 2 Ccnd1 alleles was investigated in MCL-derived cell lines and malignant cells from affected patients. The translocated Ccnd1 allele was observed significantly more distant from the nuclear membrane than its nontranslocated counterpart, with a very high proportion of IgH-Ccnd1 chromosomal segments localized next to a nucleolus. These perinucleolar areas were found to contain active RNA polymerase II (PolII) clusters. Nucleoli are rich in nucleolin, a potent transcription factor that we found to bind sites within the Ccnd1 gene specifically in MCL cells and to activate Ccnd1 transcription. We propose that the Ccnd1 transcriptional activation in MCL cells relates to the repositioning of the rearranged IgH-Ccnd1-carrying chromosomal segment in a nuclear territory with abundant nucleolin and active PolII molecules. Similar transforming events could occur in Burkitt and other B-cell lymphomas. PMID:24452204

  13. Ischemia/reperfusion activates myocardial innate immune response: the key role of the toll-like receptor

    PubMed Central

    Vilahur, Gemma; Badimon, Lina

    2014-01-01

    Recent data have indicated that the myocardium may act as an immune organ initiating cardiac innate immune response and inflammation. It has been suggested that activation of the immune system occurs upon the interaction of damage-associated molecular patterns (DAMPs) generated and released during ischemic damage with pattern recognition receptors (Toll like receptors; TLR) present in cardiac cells. Among TLRs, TLR4, and TLR2 are the ones mostly expressed in cardiac tissue. Whereas TLR4 has shown to play a detrimental role in myocardial ischemia/reperfusion (I/R) injury, the effect elicited by TLR2 activation remains controversial. Once activated, TLR signaling may occur via the Myd88- and Trif- dependent pathways leading to NFκB and IFN-3 activation, respectively, and subsequent stimulation of pro-inflammatory and immunomodulatory cytokine gene expression. Cytokine release contributes to neutrophils activation, recruitment, adhesion and infiltration to the site of cardiac injury further perpetuating the inflammatory process. This mini-review will focus on the current knowledge regarding the role of the heart in inducing and coordinating the innate inflammatory response via the TLR signaling pathway in myocardial I/R injury. PMID:25566092

  14. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. PMID:25449264

  15. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  16. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  17. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation.

    PubMed

    Martinez, Emily M; Yoshida, Miya C; Candelario, Tara Lynne T; Hughes-Fulford, Millie

    2015-03-15

    Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders. PMID:25568077

  18. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation

    PubMed Central

    Martinez, Emily M.; Yoshida, Miya C.; Candelario, Tara Lynne T.

    2015-01-01

    Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders. PMID:25568077

  19. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  20. The hydrogen chemistry of the FeMo-co active site of nitrogenase.

    PubMed

    Dance, Ian

    2005-08-10

    The chemical mechanism by which nitrogenase enzymes catalyze the hydrogenation of N(2) (and other multiply bonded substrates) at the N(c)Fe(7)MoS(9)(homocitrate) active site (FeMo-co) is unknown, despite the accumulation of much data on enzyme reactivity and the influences of key amino acids surrounding FeMo-co. The mutual influences of H(2), substrates, and the inhibitor CO on reactivity are key experimental tests for postulated mechanisms. Fundamental to all aspects of mechanism is the accumulation of H atoms (from e(-) + H(+)) on FeMo-co, and the generation and influences of coordinated H(2). Here, I argue that the first introduction of H is via a water chain terminating at water 679 (PDB structure , Azotobacter vinelandii) to one of the mu(3)-S atoms (S3B) of FeMo-co. Next, using validated density functional calculations of a full chemical representation of FeMo-co and its connected residues (alpha-275(Cys), alpha-442(His)), I have characterized more than 80 possibilities for the coordination of up to three H atoms, and H(2), and H + H(2), on the S2A, Fe2, S2B, Fe6, S3B domain of FeMo-co, which is favored by recent targeted mutagenesis results. Included are calculated reaction profiles for movements of H atoms (between S and Fe, and between Fe and Fe), for the generation of Fe-H(2), for association and dissociation of Fe-H(2) at various reduction levels, and for H/H(2) exchange. This is new hydrogen chemistry on an unprecedented coordination frame, with some similarities to established hydrogen coordination chemistry, and with unexpected and unprecedented structures such as Fe(S)(3)(H(2))(2)(H) octahedral coordination. General principles for the hydrogen chemistry of FeMo-co include (1) the stereochemical mobility of H bound to mu(3)-S, (2) the differentiated endo- and exo- positions at Fe for coordination of H and/or H(2), and (3) coordinative allosteric influences in which structural and dynamic aspects of coordination at one Fe atom are affected by

  1. Reduction of urease activity by interaction with the flap covering the active site.

    PubMed

    Macomber, Lee; Minkara, Mona S; Hausinger, Robert P; Merz, Kenneth M

    2015-02-23

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  2. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  3. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  4. Small Molecule Active Site Directed Tools for Studying Human Caspases.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S; Drag, Marcin

    2015-11-25

    Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes. PMID:26551511

  5. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  6. Structural Insights into the Catalytic Active Site and Activity of Human Nit2/ω-Amidase

    PubMed Central

    Chien, Chin-Hsiang; Gao, Quan-Ze; Cooper, Arthur J. L.; Lyu, Jyun-Hong; Sheu, Sheh-Yi

    2012-01-01

    Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/ω-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/ω-amidase was determined with α-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116–128 deletion to validate the role of key residues and the 116–128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/ω-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/ω-amidase. A catalytic triad and the loop residues 116–128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases. PMID:22674578

  7. Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.

    PubMed

    Koutsioulis, Dimitris; Lyskowski, Andrzej; Mäki, Seija; Guthrie, Ellen; Feller, Georges; Bouriotis, Vassilis; Heikinheimo, Pirkko

    2010-01-01

    Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology. PMID:19916164

  8. Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays.

    PubMed

    Liu, Ji-Yuan; Chen, Xi-En; Zhang, Ya-Lin

    2015-01-01

    Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs. PMID:26190207

  9. Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays

    PubMed Central

    Liu, Ji-Yuan; Chen, Xi-En; Zhang, Ya-Lin

    2015-01-01

    Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs. PMID:26190207

  10. School Pharmacist/School Environmental Hygienic Activities at School Site.

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  11. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  12. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  13. Screening Active Components from Yu-Ping-Feng-San for Regulating Initiative Key Factors in Allergic Sensitization

    PubMed Central

    Zhu, Zhijie; Yu, Xi; Liu, Hailiang; Wang, Huizhu; Fan, Hongwei; Wang, Dawei; Jiang, Guorong; Hong, Min

    2014-01-01

    Yu-ping-feng-san (YPFS) is a Chinese medical formula that is used clinically for allergic diseases and characterized by reducing allergy relapse. Our previous studies demonstrated that YPFS efficiently inhibited T helper 2 cytokines in allergic inflammation. The underlying mechanisms of action of YPFS and its effective components remain unclear. In this study, it was shown that YPFS significantly inhibited production of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived initiative factor in allergic inflammation, in vitro and in vivo. A method of human bronchial epithelial cell (16HBE) binding combined with HPLC-MS (named 16HBE-HPLC-MS) was established to explore potential active components of YPFS. The following five components bound to 16HBE cells: calycosin-7-glucoside, ononin, claycosin, sec-o-glucosylhamaudol and formononetin. Serum from YPFS-treated mice was analyzed and three major components were detected claycosin, formononetin and cimifugin. Among these, claycosin and formononetin were detected by 16HBE-HPLC-MS and in the serum of YPFS-treated mice. Claycosin and formononetin decreased the level of TSLP markedly at the initial stage of allergic inflammation in vivo. Nuclear factor (NF)-κB, a key transcription factor in TSLP production, was also inhibited by claycosin and formononetin, either in terms of transcriptional activation or its nuclear translocation in vitro. Allergic inflammation was reduced by claycosin and formononetin when they are administered only at the initial stage in a murine model of atopic contact dermatitis. Thus, epithelial cell binding combined with HPLC-MS is a valid method for screening active components from complex mixtures of Chinese medicine. It was demonstrated that the compounds screened from YPFS significantly attenuated allergic inflammation probably by reducing TSLP production via regulating NF-κB activation. PMID:25198676

  14. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  15. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  16. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  17. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  18. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  19. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  20. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ...BOEM is reopening the comment period announced in the Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North...

  1. Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry*

    PubMed Central

    Song, Hongjian; Olsen, Ole H.; Persson, Egon; Rand, Kasper D.

    2014-01-01

    Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease. PMID:25344622

  2. A Unique Combination of Nutritionally Active Ingredients Can Prevent Several Key Processes Associated with Atherosclerosis In Vitro

    PubMed Central

    Moss, Joe W. E.; Davies, Thomas S.; Garaiova, Iveta; Plummer, Sue F.; Michael, Daryn R.; Ramji, Dipak P.

    2016-01-01

    Introduction Atherosclerosis is the underlying cause of cardiovascular disease that leads to more global mortalities each year than any other ailment. Consumption of active food ingredients such as phytosterols, omega-3 polyunsaturated fatty acids and flavanols are known to impart beneficial effects on cardiovascular disease although the combined actions of such agents in atherosclerosis is poorly understood. The aim of this study was to screen a nutritional supplement containing each of these active components for its anti-atherosclerotic effect on macrophages in vitro. Results The supplement attenuated the expression of intercellular adhesion molecule-1 and macrophage chemoattractant protein-1 in human and murine macrophages at physiologically relevant doses. The migratory capacity of human monocytes was also hindered, possibly mediated by eicosapentaenoic acid and catechin, while the ability of foam cells to efflux cholesterol was improved. The polarisation of murine macrophages towards a pro-inflammatory phenotype was also attenuated by the supplement. Conclusion The formulation was able to hinder multiple key steps of atherosclerosis development in vitro by inhibiting monocyte recruitment, foam cell formation and macrophage polarisation towards an inflammatory phenotype. This is the first time a combination these ingredients has been shown to elicit such effects and supports its further study in preclinical in vivo models. PMID:26950833

  3. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

  4. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  5. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

  6. Structure of Arabidopsis thaliana 5-methylthioribose Kinase Reveals a More Occluded Active Site Than its Bacterial Homolog

    SciTech Connect

    Ku,S.; Cornell, K.; Howell, P.

    2007-01-01

    Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATP?S and MTR has been determined at 1.9 Angstroms resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATP?S analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

  7. Active sites for NO reduction over Fe-ZSM-5 catalysts.

    PubMed

    Schwidder, M; Santhosh Kumar, M; Brückner, A; Grünert, W

    2005-02-14

    A study of Fe-ZSM-5 catalysts with variable amounts of isolated, oligomeric and heavily aggregated Fe3+ oxo sites (as evidenced by UV-Vis and EPR spectroscopic data) and their catalytic properties in the selective catalytic reduction of NO by isobutane or by NH3 is presented, which allows development of a unified concept of the active Fe sites in these reactions, according to which isolated Fe sites catalyse both SCR reactions while oligomeric sites, though also involved in the selective reduction path, limit the catalyst performance by causing the total oxidation of the reductant. PMID:15685345

  8. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  9. From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

    PubMed Central

    Klimacek, Mario; Nidetzky, Bernd

    2010-01-01

    Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 105-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn→Asp substitutions behaved as a slow “fructose reductase” at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8–10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively “one-way” reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. PMID:20639204

  10. Structure–activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation

    PubMed Central

    Hay, Debbie L; Harris, Paul WR; Kowalczyk, Renata; Brimble, Margaret A; Rathbone, Dan L; Barwell, James; Conner, Alex C; Poyner, David R

    2014-01-01

    Background and Purpose: The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1–7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3–6 and 8–9, excluding Cys-2 and Cys-7. Experimental Approach: CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key Results: Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and Implications: Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1–7 ring also contribute to agonist activity. PMID:24125506

  11. Mechanistic and Bioinformatic Investigation of a Conserved Active Site Helix in α-Isopropylmalate Synthase from Mycobacterium tuberculosis, a Member of the DRE-TIM Metallolyase Superfamily

    PubMed Central

    2015-01-01

    The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily. PMID:24720347

  12. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    PubMed Central

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B; Sagi, Irit; Havenith, Martina

    2012-01-01

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site. PMID:21926991

  13. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  14. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

  15. Msn2p/Msn4p act as a key transcriptional activator of yeast cytoplasmic thiol peroxidase II.

    PubMed

    Hong, Seung-Keun; Cha, Mee-Kyung; Choi, Yong-Soo; Kim, Won-Cheol; Kim, Il-Han

    2002-04-01

    We observed that the transcription of Saccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of the cTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion of Tor1, Tor2, Ras1, and Ras2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription of cTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells

  16. Calculation of Vibrational Shifts of Nitrile Probes in the Active Site of Ketosteroid Isomerase upon Ligand Binding

    PubMed Central

    Layfield, Joshua P.

    2012-01-01

    The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analog equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active site water molecule that is directly hydrogen bonded to the nitrile probe, resulting in a more linear CNH angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis. PMID:23210919

  17. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    SciTech Connect

    Carra,J.; McHugh, C.; Mulligan, S.; Machiesky, L.; Soares, A.; Millard, C.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

  18. Assessment of the site of ventricular activation by Fourier analysis of gated blood-pool studies

    SciTech Connect

    Links, J.M.; Raichlen, J.S.; Wagner, H.N. Jr.; Reid, P.R.

    1985-01-01

    The authors studied the use of first-harmonic Fourier analysis of gated blood-pool images to assess the site of ventricular activation in a group of 12 patients undergoing electrophysiologic pacing studies. They acquired gated blood-pool studies during pacing at up to four sites at each of two different rates. A total of 50 studies were made. At a pacing rate of 100 beats/min, when the pacing electrode was the right-ventricular outflow tract, 7/8; at the anterolateral left-ventricular wall, 4/4. When the Fourier activation site was at the right-ventricular apex, 9/9 times the pacing electrode was there; at the right-ventricular outflow tract, 7/10; in the left ventricle, 4/4. Fourier analysis of gated blood-pool studies can help identify the site of ventricular activation but is not sufficiently accurate to fully replace endocardial mapping.

  19. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  20. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  1. NMR Localization of Divalent Cations at the Active Site of the Neurospora VS Ribozyme Provides Insights into RNA–Metal-Ion Interactions

    PubMed Central

    2013-01-01

    Metal cations represent key elements of RNA structure and function. In the Neurospora VS ribozyme, metal cations play diverse roles; they are important for substrate recognition, formation of the active site, and shifting the pKa’s of two key nucleobases that contribute to the general acid–base mechanism. Recently, we determined the NMR structure of the A730 loop of the VS ribozyme active site (SLVI) that contributes the general acid (A756) in the enzymatic mechanism of the cleavage reaction. Our studies showed that magnesium (Mg2+) ions are essential to stabilize the formation of the S-turn motif within the A730 loop that exposes the A756 nucleobase for catalysis. In this article, we extend these NMR investigations by precisely mapping the Mg2+-ion binding sites using manganese-induced paramagnetic relaxation enhancement and cadmium-induced chemical-shift perturbation of phosphorothioate RNAs. These experiments identify five Mg2+-ion binding sites within SLVI. Four Mg2+ ions in SLVI are associated with known RNA structural motifs, including the G–U wobble pair and the GNRA tetraloop, and our studies reveal novel insights about Mg2+ ion binding to these RNA motifs. Interestingly, one Mg2+ ion is specifically associated with the S-turn motif, confirming its structural role in the folding of the A730 loop. This Mg2+ ion is likely important for formation of the active site and may play an indirect role in catalysis. PMID:24364590

  2. Waste-Activated Sludge Fermentation for Polyacrylamide Biodegradation Improved by Anaerobic Hydrolysis and Key Microorganisms Involved in Biological Polyacrylamide Removal

    PubMed Central

    Dai, Xiaohu; Luo, Fan; Zhang, Dong; Dai, Lingling; Chen, Yinguang; Dong, Bin

    2015-01-01

    During the anaerobic digestion of dewatered sludge, polyacrylamide (PAM), a chemical conditioner, can usually be consumed as a carbon and nitrogen source along with other organic matter (e.g., proteins and carbohydrates in the sludge). However, a significant accumulation of acrylamide monomers (AMs) was observed during the PAM biodegradation process. To improve the anaerobic hydrolysis of PAM, especially the amide hydrolysis process, and to avoid the generation of the intermediate product AM, a new strategy is reported herein that uses an initial pH of 9, 200 mg COD/L of PAM and a fermentation time of 17 d. First, response surface methodology (RSM) was applied to optimize PAM removal in the anaerobic digestion of the sludge. The biological hydrolysis of PAM reached 86.64% under the optimal conditions obtained from the RSM. Then, the mechanisms for the optimized parameters that significantly improved the biological hydrolysis of PAM were investigated by the synergistic effect of the main organic compounds in the sludge, the floc size distribution, and the enzymatic activities. Finally, semi-continuous-flow experiments for a microbial community study were investigated based on the determination of key microorganisms involved in the biological hydrolysis of PAM. PMID:26144551

  3. Waste-Activated Sludge Fermentation for Polyacrylamide Biodegradation Improved by Anaerobic Hydrolysis and Key Microorganisms Involved in Biological Polyacrylamide Removal.

    PubMed

    Dai, Xiaohu; Luo, Fan; Zhang, Dong; Dai, Lingling; Chen, Yinguang; Dong, Bin

    2015-01-01

    During the anaerobic digestion of dewatered sludge, polyacrylamide (PAM), a chemical conditioner, can usually be consumed as a carbon and nitrogen source along with other organic matter (e.g., proteins and carbohydrates in the sludge). However, a significant accumulation of acrylamide monomers (AMs) was observed during the PAM biodegradation process. To improve the anaerobic hydrolysis of PAM, especially the amide hydrolysis process, and to avoid the generation of the intermediate product AM, a new strategy is reported herein that uses an initial pH of 9, 200 mg COD/L of PAM and a fermentation time of 17 d. First, response surface methodology (RSM) was applied to optimize PAM removal in the anaerobic digestion of the sludge. The biological hydrolysis of PAM reached 86.64% under the optimal conditions obtained from the RSM. Then, the mechanisms for the optimized parameters that significantly improved the biological hydrolysis of PAM were investigated by the synergistic effect of the main organic compounds in the sludge, the floc size distribution, and the enzymatic activities. Finally, semi-continuous-flow experiments for a microbial community study were investigated based on the determination of key microorganisms involved in the biological hydrolysis of PAM. PMID:26144551

  4. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.

    PubMed

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-04-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a(w). The apparent turnover number k(cat)(app) in these systems ranged from 0.2 to 80 s(-1) and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a(w). (19)F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a(w). The rates of NMR-derived fast (k > 10(7) s(-1)) and slow (k < 10(4) s(-1)) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k(cat). The (19)F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant epsilon(as), which increased with hydration to epsilon(as) approximately 13 in each solvent. In both solvents, the transition state free energy data and epsilon(as) followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  5. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  6. On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase.

    PubMed

    Bustos-Jaimes, Ismael; Sosa-Peinado, Alejandro; Rudiño-Piñera, Enrique; Horjales, Eduardo; Calcagno, Mario L

    2002-05-24

    The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state. PMID

  7. Identification of the conserved spatial position of key active-site atoms in glycoside hydrolase 13 family members.

    PubMed

    Kumar, Vikash

    2010-07-19

    A computational study on the glycoside hydrolase 13 (GH13) family of the CAZy database has been carried out at the atomic level in order to identify the conserved positions that may be responsible for recognition of the substrate. Analysis with substrate analog-, inhibitor-, or product-bound 3D structures was carried out to find the atomic spatial arrangement of the amino acids that make -2, -1, +1, and +2 subsites and water oxygen atoms around the ligand. The identified conserved positions of subsites were independent from the nature of the amino acid. The -1 and +1 subsites have more conserved positions than the -2 and +2 subsites. Some of the clusters of the -1 and +1 subsites have atoms of the same chemical nature. A spatially conserved position for water, which is stabilized by a hydrogen bond with the carboxyl group of a proton donor (Glu) and Asp of the catalytic triad, was found in the -1 subsite of 75% of the enzymes subjected to analysis. This position could be the region of hydrolytic water. PMID:20557875

  8. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

  9. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  10. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  11. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  12. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  13. Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

  14. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

  15. Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica.

    PubMed

    Kuettner, E Bartholomeus; Keim, Antje; Kircher, Markus; Rosmus, Susann; Sträter, Norbert

    2008-03-21

    Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 A resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two alpha/beta domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30 degrees from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity. PMID:18258259

  16. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  17. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. PMID:27285815

  18. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  19. The conserved active-site loop residues of ferrochelatase induce porphyrin conformational changes necessary for catalysis.

    SciTech Connect

    Haddad, Raid Edward; Shelnutt, John Allen; Shi, Zhen; Ferreira, Gloria C.; Franco, Ricardo T.

    2005-05-01

    Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode {gamma}{sub 15}, a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.

  20. Starting Right: Using "Biophilia," Organism Cards, & Key Themes in Biology to Introduce Student-Centered Active-Learning Strategies at the Beginning of a Course

    ERIC Educational Resources Information Center

    Metzger, Kelsey

    2013-01-01

    To create rich learning experiences, it is important to engage students from the very beginning of a course and lay the foundation for constructing a community of active learners. The activities described here using "organism cards" connect students' previous knowledge to course goals and address key themes in biology while initiating…

  1. Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

    PubMed Central

    Key, Michelle L.; Hajjawi, Mark O.R.; Millán, José L.; Arnett, Timothy R.

    2015-01-01

    Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1–6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5‐fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions. J. Cell. Physiol. 230: 3049–3056, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc

  2. Localization of the binding site of tissue-type plasminogen activator to fibrin.

    PubMed Central

    Ichinose, A; Takio, K; Fujikawa, K

    1986-01-01

    Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment. Images PMID:3088041

  3. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  4. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds. PMID:21877154

  5. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

    PubMed Central

    Rinehart, Jesse; Maksimova, Yelena D.; Tanis, Jessica E.; Stone, Kathryn L.; Hodson, Caleb A.; Zhang, Junhui; Risinger, Mary; Pan, Weijun; Wu, Dianqing; Colangelo, Christopher M.; Forbush, Biff; Joiner, Clinton H.; Gulcicek, Erol E.; Gallagher, Patrick G.; Lifton, Richard P.

    2010-01-01

    Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function. PMID:19665974

  6. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO

    NASA Astrophysics Data System (ADS)

    Alfonso, Dominic R.; Kauffman, Douglas; Matranga, Christopher

    2016-05-01

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25- nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts.

  7. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO.

    PubMed

    Alfonso, Dominic R; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25 (-) nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts. PMID:27179498

  8. Is active management the key to the conservation of saproxylic biodiversity? Pollarding promotes the formation of tree hollows.

    PubMed

    Sebek, Pavel; Altman, Jan; Platek, Michal; Cizek, Lukas

    2013-01-01

    Trees with hollows are key features sustaining biodiversity in wooded landscapes. They host rich assemblages of often highly specialised organisms. Hollow trees, however, have become rare and localised in Europe. Many of the associated biota is thus declining or endangered. The challenge of its conservation, therefore, is to safeguard the presence of hollow trees in sufficient numbers. Populations of numerous species associated with tree hollows and dead wood are often found in habitats that were formed by formerly common traditional silvicultural practices such as coppicing, pollarding or pasture. Although it has been occasionally mentioned that such practices increase the formation of hollows and the availability of often sun-exposed dead wood, their effect has never been quantified. Our study examined the hollow incidence in pollard and non-pollard (unmanaged) willows and the effect of pollarding on incremental growth rate by tree ring analysis. The probability of hollow occurrence was substantially higher in pollard than in non-pollard trees. Young pollards, especially, form hollows much more often than non-pollards; for instance, in trees of 50 cm DBH, the probability of hollow ocurrence was ∼0.75 in pollards, but only ∼0.3 in non-pollards. No difference in growth rate was found. Pollarding thus leads to the rapid formation of tree hollows, a habitat usually associated with old trees. It is therefore potentially a very important tool in the restoration of saproxylic habitats and conservation of hollow-dependent fauna. If applied along e.g. roads and watercourses, pollarding could also be used to increase landscape connectivity for saproxylic organisms. In reserves where pollarding was formerly practiced, its restoration would be necessary to prevent loss of saproxylic biodiversity. Our results point to the importance of active management measures for maintaining availability, and spatial and temporal continuity of deadwood microhabitats. PMID:23544142

  9. Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

    PubMed Central

    Milgrom, Y M; Murataliev, M B; Boyer, P D

    1998-01-01

    Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid. PMID:9480927

  10. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    SciTech Connect

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M.

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiOx domains are titrated on TiOx–SiO2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiOx, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H2O2 activation occurs. We use this method to determine the active site densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h-1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.

  11. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  12. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  13. Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

    Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants. PMID:8606154

  14. Addressing key concepts in physical geography through interactive learning activities in an online geo-ICT environment

    NASA Astrophysics Data System (ADS)

    Verstraeten, Gert; Steegen, An; Martens, Lotte

    2016-04-01

    The increasing number of geospatial datasets and free online geo-ICT tools offers new opportunities for education in Earth Sciences. Geospatial technology indeed provides an environment through which interactive learning can be introduced in Earth Sciences curricula. However, the effectiveness of such e-learning approaches in terms of learning outcomes has rarely been addressed. Here, we present our experience with the implementation of digital interactive learning activities within an introductory Physical Geography course attended by 90 undergraduate students in Geography, Geology, Biology and Archaeology. Two traditional lectures were replaced by interactive sessions (each 2 h) in a flexible classroom where students had to work both in team and individually in order to explore some key concepts through the integrated use of geospatial data within Google EarthTM. A first interactive lesson dealt with the classification of river systems and aimed to examine the conditions under which rivers tend to meander or to develop a braided pattern. Students were required to collect properties of rivers (river channel pattern, channel slope, climate, discharge, lithology, vegetation, etc). All these data are available on a global scale and have been added as separate map layers in Google EarthTM. Each student collected data for at least two rivers and added this information to a Google Drive Spreadsheet accessible to the entire group. This resulted in a database of more than one hundred rivers spread over various environments worldwide. In a second phase small groups of students discussed the potential relationships between river channel pattern and its controlling factors. Afterwards, the findings of each discussion group were presented to the entire audience. The same set-up was followed in a second interactive session to explore spatial variations in ecosystem properties such as net primary production and soil carbon content. The qualitative evaluation of both interactive

  15. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents

    PubMed Central

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-01-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, aw. The apparent turnover number kcatapp in these systems ranged from 0.2 to 80 s−1 and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing aw. 19F-NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on aw. The rates of NMR-derived fast (k > 107 s−1) and slow (k < 104 s−1) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with kcat. The 19F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant εas, which increased with hydration to εas ≈ 13 in each solvent. In both solvents the transition state free energy data and εas followed Kirkwood’s model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site’s electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  16. Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

    PubMed

    Scaloni, A; Jones, W M; Barra, D; Pospischil, M; Sassa, S; Popowicz, A; Manning, L R; Schneewind, O; Manning, J M

    1992-02-25

    Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions. PMID:1740429

  17. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

  18. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    PubMed

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide. PMID:26535916

  19. Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

    PubMed Central

    Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

    2014-01-01

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

  20. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  1. Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

    PubMed Central

    Wang, Lu; Fried, Stephen D.; Boxer, Steven G.; Markland, Thomas E.

    2014-01-01

    Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds. PMID:25503367

  2. Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site

    PubMed Central

    Stafford, Kate A.; Palmer III, Arthur G.

    2014-01-01

    Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold. PMID:25075292

  3. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

  4. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  5. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  6. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  7. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    SciTech Connect

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  8. Activity-dependent labeling of oxygenase enzymes in a trichloroethene-contaminated groundwater site.

    PubMed

    Lee, M Hope; Clingenpeel, Scott C; Leiser, Owen P; Wymore, Ryan A; Sorenson, Kent S; Watwood, Mary E

    2008-05-01

    A variety of naturally occurring bacteria produce enzymes that cometabolically degrade trichloroethene (TCE), including organisms with aerobic oxygenases. Groundwater contaminated with TCE was collected from the aerobic region of the Test Area North site of the Idaho National Laboratory. Samples were evaluated with enzyme activity probes, and resulted in measurable detection of toluene oxygenase activity (6-79% of the total microbial cells). Wells from both inside and outside contaminated plume showed activity. Toluene oxygenase-specific PCR primers determined that toluene-degrading genes were present in all groundwater samples evaluated. In addition, bacterial isolates were obtained and possessed toluene oxygenase enzymes, demonstrated activity, and were dominated by the phylotype Pseudomonas. This study demonstrated, through the use of enzymatic probes and oxygenase gene identification, that indigenous microorganisms at a contaminated site were cometabolically active. Documentation such as this can be used to substantiate observations of natural attenuation of TCE-contaminated groundwater plumes. PMID:17904715

  9. Cooperativity between Al Sites Promotes Hydrogen Transfer and Carbon–Carbon Bond Formation upon Dimethyl Ether Activation on Alumina

    PubMed Central

    2015-01-01

    The methanol-to-olefin (MTO) process allows the conversion of methanol/dimethyl ether into olefins on acidic zeolites via the so-called hydrocarbon pool mechanism. However, the site and mechanism of formation of the first carbon–carbon bond are still a matter of debate. Here, we show that the Lewis acidic Al sites on the 110 facet of γ-Al2O3 can readily activate dimethyl ether to yield CH4, alkenes, and surface formate species according to spectroscopic studies combined with a computational approach. The carbon–carbon forming step as well as the formation of methane and surface formate involves a transient oxonium ion intermediate, generated by a hydrogen transfer between surface methoxy species and coordinated methanol on adjacent Al sites. These results indicate that extra framework Al centers in acidic zeolites, which are associated with alumina, can play a key role in the formation of the first carbon–carbon bond, the initiation step of the industrial MTO process. PMID:27162986

  10. Cooperativity between Al Sites Promotes Hydrogen Transfer and Carbon-Carbon Bond Formation upon Dimethyl Ether Activation on Alumina.

    PubMed

    Comas-Vives, Aleix; Valla, Maxence; Copéret, Christophe; Sautet, Philippe

    2015-09-23

    The methanol-to-olefin (MTO) process allows the conversion of methanol/dimethyl ether into olefins on acidic zeolites via the so-called hydrocarbon pool mechanism. However, the site and mechanism of formation of the first carbon-carbon bond are still a matter of debate. Here, we show that the Lewis acidic Al sites on the 110 facet of γ-Al2O3 can readily activate dimethyl ether to yield CH4, alkenes, and surface formate species according to spectroscopic studies combined with a computational approach. The carbon-carbon forming step as well as the formation of methane and surface formate involves a transient oxonium ion intermediate, generated by a hydrogen transfer between surface methoxy species and coordinated methanol on adjacent Al sites. These results indicate that extra framework Al centers in acidic zeolites, which are associated with alumina, can play a key role in the formation of the first carbon-carbon bond, the initiation step of the industrial MTO process. PMID:27162986

  11. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

  12. Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

    SciTech Connect

    Fitzner, R.E.; Weiss, S.G.; Stegen, J.A.

    1994-06-01

    The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

  13. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

  14. The active site of low-temperature methane hydroxylation in iron-containing zeolites.

    PubMed

    Snyder, Benjamin E R; Vanelderen, Pieter; Bols, Max L; Hallaert, Simon D; Böttger, Lars H; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2016-08-18

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(ii), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species-α-Fe(ii) and α-O-are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive 'spectator' iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(ii) to be a mononuclear, high-spin, square planar Fe(ii) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(iv)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function-producing what is known in the context of metalloenzymes as an 'entatic' state-might be a useful way to tune the activity of heterogeneous catalysts. PMID:27535535

  15. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  16. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  17. Monitoring of geological activity on astronomical sites of the Canary Islands, Hawaii, and Chile

    NASA Astrophysics Data System (ADS)

    Eff-Darwich, Antonio; Garcia-Lorenzo, Begoña; Rodriguez-Losada, Jose A.; Hernández-Gutiérrez, Luis E.; de la Nuez, Julio; Romero-Ruiz, Maria C.

    2009-09-01

    Future large and extremely large ground-based telescopes will demand stable geological settings.Remote sensing could be an unvaluable tool to analyse the impact of geological activity at selected astronomical sites, namely the observatories of El Teide (Tenerife, Canary Islands), Roque de los Muchachos (La Palma, Canary Islands), Mauna Kea (Hawaii) and Paranal (Chile; the candidate site of Cerro Ventarrones, Chile). In this sense, the extent of lava flows, eruptive clouds or ground deformation associated to seismic and/or volcanic activity could be analysed and characterised through remote sensing.

  18. Possible peroxidase active site environment in amyloidogenic proteins: Native monomer or misfolded-oligomer; which one is susceptible to the enzymatic activity, with contribution of heme?

    PubMed

    Khodarahmi, Reza; Ashrafi-Kooshk, Mohammad Reza; Khodarahmi, Sina; Ghadami, Seyyed Abolghasem; Mostafaie, Ali

    2015-09-01

    Amyloid states of many proteins complex with heme and exhibit significant non-specific peroxidase activity, compared to free heme. Neurotransmitter deficiency, generation of neurotoxins, altered activity/metabolism of key enzymes and cellular DNA damage are possible evidences highlighting the importance of the uncontrollable peroxidase activity in Alzheimer's disease (AD)-involved brain cells. Despite extensive experimental work was carried out on this field, discrepancy on chronological precedence of amyloid aggregation and oxidative reactions as well as the mechanism involved in the peroxidase-induced oxidative stress is still not completely understood. In this study, we highlight further that heme cofactor readily complexes with structural intermediates of amyloid aggregates of ovalbumin, lactoglobulin and crystallin and report the ability of "heme-amyloid aggregate/oligomer") to produce peroxidase-like active site. Histidine side chains are also proposed as both distal and proximal residues required for proper function of these peroxidase systems. Taking uncontrollable peroxidase activity of "Aβ-heme" complex into account, it appears that this process, as a new opened dimension in AD pathologic research, provides structural/mechanistic basis for more efficient therapeutic strategies against neurodegenerative diseases. PMID:26123814

  19. Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase.

    PubMed

    Pekkala, Satu; Martínez, Ana I; Barcelona, Belén; Gallego, José; Bendala, Elena; Yefimenko, Igor; Rubio, Vicente; Cervera, Javier

    2009-12-01

    NAG (N-acetyl-L-glutamate), the essential allosteric activator of the first urea cycle enzyme, CPSI (carbamoyl phosphate synthetase I), is a key regulator of this crucial cycle for ammonia detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed identification of the NAG site in the crystal structure of human CPSI C-terminal domain. The site, a pocket lined by invariant residues and located between the central beta-sheet and two alpha-helices, opens at the beta-sheet C-edge and is roofed by a three-residue lid. It can tightly accommodate one extended NAG molecule having the delta-COO- at the pocket entry, the alpha-COO- and acetamido groups tightly hydrogen bonded to the pocket, and the terminal methyl of the acetamido substituent surrounded by hydrophobic residues. This binding mode is supported by the observation of reduced NAG affinity upon mutation of NAG-interacting residues of CPSI (recombinantly expressed using baculovirus/insect cells); by the fine-mapping of the N-chloroacetyl-L-glutamate photoaffinity labelling site of CPSI; and by previously established structure-activity relationships for NAG analogues. The location of the NAG site is identical to that of the weak bacterial CPS activator IMP (inosine monophosphate) in Escherichia coli CPS, indicating a common origin for these sites and excluding any relatedness to the binding site of the other bacterial CPS activator, ornithine. Our findings open the way to the identification of CPSI deficiency patients carrying NAG site mutations, and to the possibility of tailoring the activator to fit a given NAG site mutation, as exemplified here with N-acetyl-L(+/-)-beta-phenylglutamate for the W1410K CPSI mutation. PMID:19754428

  20. Is the protein surrounding the active site critical for hydrogen peroxide reduction by selenoprotein glutathione peroxidase? An ONIOM study.

    PubMed

    Prabhakar, Rajeev; Vreven, Thom; Frisch, Michael J; Morokuma, Keiji; Musaev, Djamaladdin G

    2006-07-13

    In this ONIOM(QM:MM) study, we evaluate the role of the protein surroundings in the mechanism of H2O2 reduction catalyzed by the glutathione peroxidase enzyme, using the whole monomer (3113 atoms in 196 amino acid residues) as a model. A new optimization scheme that allows the full optimization of transition states for large systems has been utilized. It was found that in the presence of the surrounding protein the optimized active site structure bears a closer resemblance to the one in the X-ray structure than that without the surrounding protein. H2O2 reduction occurs through a two-step mechanism. In the first step, the selenolate anion (E-Se(-)) formation occurs with a barrier of 16.4 kcal/mol and is endothermic by 12.0 kcal/mol. The Gln83 residue plays the key role of the proton abstractor, which is in line with the experimental suggestion. In the second step, the O-O bond is cleaved, and selenenic acid (R-Se-OH) and a water molecule are formed. The calculated barrier for this process is 6.0 kcal/mol, and it is exothermic by 80.9 kcal/mol. The overall barrier of 18.0 kcal/mol for H2O2 reduction is in reasonable agreement with the experimentally measured barrier of 14.9 kcal/mol. The protein surroundings has been calculated to exert a net effect of only 0.70 kcal/mol (in comparison to the "active site only" model including solvent effects) on the overall barrier, which is most likely due to the active site being located at the enzyme surface. PMID:16821888

  1. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  2. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  3. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

  4. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    NASA Astrophysics Data System (ADS)

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S.; Drakou, Evangelia G.; Pantis, John D.

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  5. Small activating RNA binds to the genomic target site in a seed-region-dependent manner

    PubMed Central

    Meng, Xing; Jiang, Qian; Chang, Nannan; Wang, Xiaoxia; Liu, Chujun; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2016-01-01

    RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition. PMID:26873922

  6. A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization†

    PubMed Central

    Uzun, Ozcan; Gabriel, Abram

    2001-01-01

    Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme. PMID:11413300

  7. DNA binding induces active site conformational change in the human TREX2 3'-exonuclease.

    PubMed

    de Silva, Udesh; Perrino, Fred W; Hollis, Thomas

    2009-04-01

    The TREX enzymes process DNA as the major 3'-->5' exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3' hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site. PMID:19321497

  8. Subsite-specific contributions of different aromatic residues in the active site architecture of glycoside hydrolase family 12

    PubMed Central

    Zhang, Xiaomei; Wang, Shuai; Wu, Xiuyun; Liu, Shijia; Li, Dandan; Xu, Hao; Gao, Peiji; Chen, Guanjun; Wang, Lushan

    2015-01-01

    The active site architecture of glycoside hydrolase (GH) is a contiguous subregion of the enzyme constituted by residues clustered in the three-dimensional space, recognizing the monomeric unit of ligand through hydrogen bonds and hydrophobic interactions. Mutations of the key residues in the active site architecture of the GH12 family exerted different impacts on catalytic efficiency. Binding affinities between the aromatic amino acids and carbohydrate rings were quantitatively determined by isothermal titration calorimetry (ITC) and the quantum mechanical (QM) method, showing that the binding capacity order of Tyr>Trp>His (and Phe) was determined by their side-chain properties. The results also revealed that the binding constant of a certain residue remained unchanged when altering its location, while the catalytic efficiency changed dramatically. Increased binding affinity at a relatively distant subsite, such as the mutant of W7Y at the −4 subsite, resulted in a marked increase in the intermediate product of cellotetraose and enhanced the reactivity of endoglucanase by 144%; while tighter binding near the catalytic center, i.e. W22Y at the −2 subsite, enabled the enzyme to bind and hydrolyze smaller oligosaccharides. Clarification of the specific roles of the aromatics at different subsites may pave the way for a more rational design of GHs. PMID:26670009

  9. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    NASA Astrophysics Data System (ADS)

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  10. Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.

    PubMed Central

    Kachurin, A M; Golubev, A M; Geisow, M M; Veselkina, O S; Isaeva-Ivanova, L S; Neustroev, K N

    1995-01-01

    alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide. Images Figure 10 PMID:8948456

  11. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

  12. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  13. Immobilized low-activity waste site borehole 299-E17-21

    SciTech Connect

    Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

    1998-08-01

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area.

  14. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  15. Effect of broadcast baiting on abundance patterns of red imported fire ants (Hymenoptera: Formicidae) and key local ant genera at long-term monitoring sites in Brisbane, Australia.

    PubMed

    McNaught, Melinda K; Wylie, F Ross; Harris, Evan J; Alston, Clair L; Burwell, Chris J; Jennings, Craig

    2014-08-01

    In 2001, the red imported fire ant (Solenopsis invicta Buren) was identified in Brisbane, Australia. An eradication program involving broadcast bait treatment with two insect growth regulators and a metabolic inhibitor began in September of that year and is currently ongoing. To gauge the impacts of these treatments on local ant populations, we examined long-term monitoring data and quantified abundance patterns of S. invicta and common local ant genera using a linear mixed-effects model. For S. invicta, presence in pitfalls reduced over time to zero on every site. Significantly higher numbers of S. invicta workers were collected on high-density polygyne sites, which took longer to disinfest compared with monogyne and low-density polygyne sites. For local ants, nine genus groups of the 10 most common genera analyzed either increased in abundance or showed no significant trend. Five of these genus groups were significantly less abundant at the start of monitoring on high-density polygyne sites compared with monogyne and low-density polygyne sites. The genus Pheidole significantly reduced in abundance over time, suggesting that it was affected by treatment efforts. These results demonstrate that the treatment regime used at the time successfully removed S. invicta from these sites in Brisbane, and that most local ant genera were not seriously impacted by the treatment. These results have important implications for current and future prophylactic treatment efforts, and suggest that native ants remain in treated areas to provide some biological resistance to S. invicta. PMID:25195416

  16. Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: Does a third metal binding site modulate endonuclease activity?

    PubMed Central

    Ho, Ming-Hsun; De Vivo, Marco; Peraro, Matteo Dal; Klein, Michael L.

    2010-01-01

    Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg2+ concentration. Moreover, in RNase H the glutamate residue E188 has been shown to be essential for full enzymatic activation regardless of the Mg2+ concentration. The catalytic center is known to contain two Mg2+ ions (Nowotny et al.) and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg2+. Importantly, the presence of a third Mg2+ ion, bound to the second-shell ligand E188, is persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations depending on the concentration of Mg2+. At standard concentration, a third Mg2+ is found in the catalytic pocket but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg2+ ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg2+ ions nearby the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A, and also supports the key role of E188 in localizing the third Mg2+ ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD

  17. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    PubMed

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

  18. CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways.

    PubMed

    Wang, Yang-Gang; Cantu, David C; Lee, Mal-Soon; Li, Jun; Glezakou, Vassiliki-Alexandra; Rousseau, Roger

    2016-08-24

    We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations. PMID:27480512

  19. Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

    PubMed

    Tepper, A D; Dammann, H; Bominaar, A A; Véron, M

    1994-12-23

    Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase. PMID:7798215

  20. Lessons learned from DOE site culture change activities: Implications for waste management organizations

    SciTech Connect

    Kurstedt, H.A. Jr.; Howard, E.M.; Doss, A.R.; Mallak, L.A.

    1991-01-01

    Management Systems Laboratories (MSL) has worked with the US Department of Energy (DOE) and several of its contractors as they understand and assess the DOE culture change and change the contractor culture to serve DOE's needs. Primarily, these contractors have been those whose responsibilities include starting up and operating weapons materials facilities. The number and scope of these activities have escalated and expanded to contractors at DOE sites such as Westinghouse at the Savannah River Site (SRS) in Aiken, South Carolina, EG G at the Rocky Flats Plant (RFP) in Golden, Colorado, and Westinghouse at the Feed Materials Processing Center (FMPC) in Fernald, Ohio. The point of this paper is not to compare or contrast the relative merit of one site over another. It is to show the lessons, good and bad, and use and communicate those lessons, especially those lessons transferable to other sites in similar situations. 8 refs., 1 fig.

  1. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  2. Recent Experience Using Active Love Wave Techniques to Characterize Seismographic Station Sites

    NASA Astrophysics Data System (ADS)

    Martin, A. J.; Yong, A.; Salomone, L.

    2014-12-01

    Active-source Love waves recorded by the multi-channel analysis of surface wave (MASLW) technique were recently analyzed in two site characterization projects. Between 2010 and 2011, the 2009 American Recovery and Reinvestment Act (ARRA) funded GEOVision to conduct geophysical investigations at 189 seismographic stations—185 in California and 4 in the Central Eastern U.S. (CEUS). The original project plan was to utilize active and passive Rayleigh wave-based techniques to obtain shear-wave velocity (VS) profiles to a minimum depth of 30 m and the time-averaged VS of the upper 30 meters (VS30). Early in the investigation it became evident that Rayleigh wave techniques, such as multi-channel analysis of surface waves (MASRW), were not effective at characterizing all sites. Shear-wave seismic refraction and MASLW techniques were therefore applied. The MASLW technique was deployed at a total of 38 sites, in addition to other methods, and used as the primary technique to characterize 22 sites, 5 of which were also characterized using Rayleigh wave techniques. In 2012, the Electric Power Research Institute funded characterization of 33 CEUS station sites. Based on experience from the ARRA investigation, both MASRW and MASLW data were acquired by GEOVision at 24 CEUS sites—the remaining 9 sites and 2 overlapping sites were characterized by University of Texas, Austin. Of the 24 sites characterized by GEOVision, 16 were characterized using MASLW data, 4 using both MASLW and MASRW data and 4 using MASRW data. Love wave techniques were often found to perform better, or at least yield phase velocity data that could be more readily modeled using the fundamental mode assumption, at shallow rock sites, sites with steep velocity gradients, and, sites with a thin, low velocity, surficial soil layer overlying stiffer sediments. These types of velocity structure often excite dominant higher modes in Rayleigh wave data, but not in Love wave data. At such sites, it may be possible

  3. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. PMID:20603179

  4. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

  5. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  6. A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

    PubMed Central

    Tanaka, Takeshi; Fujiwara, Shinsuke; Nishikori, Shingo; Fukui, Toshiaki; Takagi, Masahiro; Imanaka, Tadayuki

    1999-01-01

    We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. PMID:10583986

  7. Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II).

    PubMed Central

    Nic a'Bháird, N; Yankovskaya, V; Ramsay, R R

    1998-01-01

    The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1mol of histidine was modified per mol of CPT-II.When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity. The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely. It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated COT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced. Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine. PMID:9480926

  8. Identification of active sites in gold-catalyzed hydrogenation of acrolein.

    PubMed

    Mohr, Christian; Hofmeister, Herbert; Radnik, Jörg; Claus, Peter

    2003-02-19

    The active sites of supported gold catalysts, favoring the adsorption of C=O groups of acrolein and subsequent reaction to allyl alcohol, have been identified as edges of gold nanoparticles. After our recent finding that this reaction preferentially occurs on single crystalline particles rather than multiply twinned ones, this paper reports on a new approach to distinguish different features of the gold particle morphology. Elucidation of the active site issue cannot be simply done by varying the size of gold particles, since the effects of faceting and multiply twinned particles may interfere. Therefore, modification of the gold particle surface by indium has been used to vary the active site characteristics of a suitable catalyst, and a selective decoration of gold particle faces has been observed, leaving edges free. This is in contradiction to theoretical predictions, suggesting a preferred occupation of the low-coordinated edges of the gold particles. On the bimetallic catalyst, the desired allyl alcohol is the main product (selectivity 63%; temperature 593 K, total pressure p(total) = 2 MPa). From the experimentally proven correlation between surface structure and catalytic behavior, the edges of single crystalline gold particles have been identified as active sites for the preferred C=O hydrogenation. PMID:12580618

  9. Strategies and Activities for Using Local Communities as Environmental Education Sites.

    ERIC Educational Resources Information Center

    Roth, Charles E.; Lockwood, Linda G.

    Presented are over 100 environmental education activities which use the local community for a learning site and resource. These lessons are grouped under seven topical headings: (1) biological neighbors, (2) physical environs, (3) built environs, (4) social environs, (5) understanding ourselves, (6) influencing change, and (7) improvement and…

  10. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-06

    ... FR 30,616) of the EA for Issuance of Leases for Wind Resource Data Collection on the Outer... (NOA) in the Federal Register (72 FR 62,672) of the Programmatic EIS for Alternative Energy Development... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the...

  11. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Contracts for Superfund Response Actions Combining Cooperative Agreements § 35.6260 Combining...

  12. Organized Agents: Canadian Teacher Unions as Alternative Sites for Social Justice Activism

    ERIC Educational Resources Information Center

    Rottmann, Cindy

    2008-01-01

    Historically teachers' federations have been some of the major organizational sites for social justice leadership in K-12 public education. Despite this history of activism, social justice teacher unionism remains a relatively underdeveloped concept. This article merges four philosophical conceptions of social justice in education: liberal…

  13. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  14. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  15. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  16. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  17. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  18. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    ERIC Educational Resources Information Center

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  19. IN VIVO ACTIVITY OF RHOPALOSIPHUM PADI VIRUS INTERNAL RIBOSOME ENTRY SITES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transc...

  20. Cyclic silicate active site and stereochemical match for apatite nucleation on pseudowollastonite bioceramic-bone interfaces.

    PubMed

    Sahai, Nita; Anseau, Michel

    2005-10-01

    Hydroxyapatite (Ca5(PO4)3(OH)) forms on pseudowollastonite (psW) (alpha-CaSiO3) in vitro in simulated body fluid, human parotid saliva and cell-culture medium, and in vivo in implanted rat tibias. We used crystallographic constraints with ab initio molecular orbital calculations to identify the active site and reaction mechanism for heterogeneous nucleation of the earliest calcium phosphate oligomer/phase. The active site is the planar, cyclic, silicate trimer (Si3O9) on the (001) face of psW. The trimer has three silanol groups (>SiOH) arranged at 60 degrees from each other, providing a stereochemical match for O atoms bonded to Ca2+ on the (001) face of hydroxyapatite. Calcium phosphate nucleation is modeled in steps as hydrolysis of surface Ca-O bonds with leaching of Ca2+ into solution, protonation of the surface Si-O groups to form silanols, calcium sorption as an inner-sphere surface complex and, attachment of HPO4(2-). Our model explains the experimental solution and high resolution transmission electron microscopy data for epitaxial hydroxyapatite growth on psW in vitro and in vivo. We propose that the cyclic silicate trimer is the universal active site for heterogeneous, stereochemically promoted nucleation on silicate-based bioactive ceramics. A critical active site-density and a point of zero charge of the bioceramic less than physiological pH are required for bioactivity. PMID:15949543

  1. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

    PubMed

    Kleinboelting, Silke; Ramos-Espiritu, Lavoisier; Buck, Hannes; Colis, Laureen; van den Heuvel, Joop; Glickman, J Fraser; Levin, Lonny R; Buck, Jochen; Steegborn, Clemens

    2016-04-29

    The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs. PMID:26961873

  2. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    SciTech Connect

    Shia, Steven; Stamos, Jennifer; Kirchhofer, Daniel; Fan, Bin; Wu, Judy; Corpuz, Raquel T.; Santell, Lydia; Lazarus, Robert A.; Eigenbrot, Charles

    2010-07-20

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 {angstrom} resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 {angstrom} resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  3. Key Nutrients.

    ERIC Educational Resources Information Center

    Federal Extension Service (USDA), Washington, DC.

    Lessons written to help trainer agents prepare aides for work with families in the Food and Nutrition Program are presented in this booklet. The key nutrients discussed in the 10 lessons are protein, carbohydrates, fat, calcium, iron, iodine, and Vitamins A, B, C, and D. the format of each lesson is as follows: Purpose, Presentation, Application…

  4. Low temperature syntheses and reactivity of Cu2O2 active-site models.

    PubMed

    Citek, Cooper; Herres-Pawlis, Sonja; Stack, T Daniel P

    2015-08-18

    Nature's facility with dioxygen outmatches modern chemistry in the oxidation and oxygenation of materials and substrates for biosynthesis and cellular metabolism. The Earth's most abundant naturally occurring oxidant is-frankly-poorly understood and controlled, and thus underused. Copper-based enzyme metallocofactors are ubiquitous to the efficient consumption of dioxygen by all domains of life. Over the last several decades, we have joined many research groups in the study of copper- and dioxygen-dependent enzymes through close investigation of synthetically derived, small-molecule active-site analogs. Simple copper-dioxygen clusters bearing structural and spectroscopic similarity to dioxygen-activating enzymes can be probed for their fundamental geometrical, electronic, and reactive properties using the tools available to inorganic and synthetic chemistry. Our exploration of the copper-dioxygen arena has sustained product evaluation of the key dynamics and reactivity of binuclear Cu2O2 compounds. Almost exclusively operating at low temperatures, from -78 °C to solution characterization even at -125 °C, we have identified numerous compounds supported by simple and easily accessed, low molecular weight ligands-chiefly families of bidentate diamine chelates. We have found that by stripping away complexity in comparison to extended protein tertiary structures or sophisticated, multinucleating architectures, we can experimentally manipulate activated compounds and open pathways of reactivity toward exogenous substrates that both inform on and extend fundamental mechanisms of oxygenase enzymes. Our recent successes have advanced understanding of the tyrosinase enzyme, and related hemocyanin and NspF, and the copper membrane monooxygenases, specifically particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO). Tyrosinase, ubiquitously distributed throughout life, is fundamental to the copper-based oxidation of phenols and the production of chromophores

  5. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    PubMed

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. PMID:23564727

  6. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  7. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  8. Chronic activation of the low affinity site of β1-adrenoceptors stimulates haemodynamics but exacerbates pressure-overload cardiac remodelling

    PubMed Central

    Kiriazis, Helen; Tugiono, Niquita; Xu, Qi; Gao, Xiao-Ming; Jennings, Nicole L; Ming, Ziqui; Su, Yidan; Klenowski, Paul; Summers, Roger J; Kaumann, Alberto; Molenaar, Peter; Du, Xiao-Jun

    2013-01-01

    BACKGROUND AND PURPOSE The β1-adrenoceptor has at least two binding sites, high and low affinity sites (β1H and β1L, respectively), which mediate cardiostimulation. While β1H-adrenoceptor can be blocked by all clinically used β-blockers, β1L-adrenoceptor is relatively resistant to blockade. Thus, chronic β1L-adrenoceptor activation may mediate persistent cardiostimulation, despite the concurrent blockade of β1H-adrenoceptors. Hence, it is important to determine the potential significance of β1L-adrenoceptors in vivo, particularly in pathological situations. EXPERIMENTAL APPROACH C57Bl/6 male mice were used. Chronic (4 or 8 weeks) β1L-adrenoceptor activation was achieved by treatment, via osmotic mini pumps, with (-)-CGP12177 (10 mg·kg−1·day−1). Cardiac function was assessed by echocardiography and micromanometry. KEY RESULTS (-)-CGP12177 treatment of healthy mice increased heart rate and left ventricular (LV) contractility. (-)-CGP12177 treatment of mice subjected to transverse aorta constriction (TAC), during weeks 4–8 or 4–12 after TAC, led to a positive inotropic effect and exacerbated fibrogenic signalling while cardiac hypertrophy tended to be more severe. (-)-CGP12177 treatment of mice with TAC also exacerbated the myocardial expression of hypertrophic, fibrogenic and inflammatory genes compared to untreated TAC mice. Washout of (-)-CGP12177 revealed a more pronounced cardiac dysfunction after 12 weeks of TAC. CONCLUSIONS AND IMPLICATIONS β1L-adrenoceptor activation provides functional support to the heart, in both normal and pathological (pressure overload) situations. Sustained β1L-adrenoceptor activation in the diseased heart exacerbates LV remodelling and therefore may promote disease progression from compensatory hypertrophy to heart failure. PMID:23750586

  9. Putting Flow Theory into Educational Practice: The Key School's Flow Activities Room. Report to the Benton Center for Curriculum and Instruction, University of Chicago.

    ERIC Educational Resources Information Center

    Whalen, Samuel P.; Csikszentmihalyi, Mihaly

    The Key School in Indianapolis, an experimental elementary school, was founded by eight experienced teachers who believed that schools can be enjoyable as well as rigorous. This report focuses on one feature of the school, the Flow Activities Room (FAR). The development of the FAR was based on the school's commitment to the theories of multiple…

  10. Functional copper at the acetyl-CoA synthase active site

    PubMed Central

    Seravalli, Javier; Gu, Weiwei; Tam, Annie; Strauss, Erick; Begley, Tadhg P.; Cramer, Stephen P.; Ragsdale, Stephen W.

    2003-01-01

    The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood–Ljungdahl pathway of autotrophic CO2 fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the “NiFeC” EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)–S3 site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu–S bond length of 2.25 Å and a metal neighbor at 2.65 Å, consistent with the Cu–Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu–S3Se environment with a 2.4-Å Se–Cu bond, strongly implicating a Cu–SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms. PMID:12589021

  11. Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.

    PubMed

    Chen, Mengbin; Chou, Wayne K W; Al-Lami, Naeemah; Faraldos, Juan A; Allemann, Rudolf K; Cane, David E; Christianson, David W

    2016-05-24

    Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule coordinates to the Mg(2+)C ion. There is no obvious role for water in the ATAS mechanism because the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg(2+)C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg(2+)C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates. PMID:27172425

  12. Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O2 Activation

    PubMed Central

    2015-01-01

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enz