Sample records for active site suggesting

  1. The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gopal, B.; Madan, Lalima L.; Betz, Stephen F.

    2010-11-10

    Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s),more » whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.« less

  2. Nature of the Active Sites for CO Reduction on Copper Nanoparticles; Suggestions for Optimizing Performance.

    PubMed

    Cheng, Tao; Xiao, Hai; Goddard, William A

    2017-08-30

    Recent experiments show that the grain boundaries (GBs) of copper nanoparticles (NPs) lead to an outstanding performance in reducing CO 2 and CO to alcohol products. We report here multiscale simulations that simulate experimental synthesis conditions to predict the structure of a 10 nm Cu NP (158 555 atoms). To identify active sites, we first predict the CO binding at a large number of sites and select four exhibiting CO binding stronger than the (211) step surface. Then, we predict the formation energy of the *OCCOH intermediate as a descriptor for C-C coupling, identifying two active sites, both of which have an under-coordinated surface square site adjacent to a subsurface stacking fault. We then propose a periodic Cu surface (4 by 4 supercell) with a similar site that substantially decreases the formation energy of *OCCOH, by 0.14 eV.

  3. De novo active sites for resurrected Precambrian enzymes

    NASA Astrophysics Data System (ADS)

    Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

    2017-07-01

    Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

  4. Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

    PubMed

    Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

    2012-01-01

    Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

  5. Non-competitive inhibition by active site binders.

    PubMed

    Blat, Yuval

    2010-06-01

    Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.

  6. 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.

  7. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    DOE PAGES

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [Cu IIOH] + species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [Cu IIOH] + active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu] 2+ and Cu 3O 3 motifs.« less

  8. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific.

    PubMed

    Thune, I; Furberg, A S

    2001-06-01

    The association between physical activity and overall and site-specific cancer risk is elaborated in relation to whether any observed dose-response association between physical activity and cancer can be interpreted in terms of how much physical activity (type, intensity, duration, frequency) is needed to influence site- and gender-specific cancer risk. Observational studies were reviewed that have examined the independent effect of the volume of occupational physical activity (OPA) and/or leisure time physical activity (LPA) on overall and site-specific cancer risk. The evidence of cohort and case-control studies suggests that both leisure time and occupational physical activity protect against overall cancer risk, with a graded dose-response association suggested in both sexes. Confounding effects such as diet, body weight, and parity are often included as a covariate in the analyses, with little influence on the observed associations. A crude graded inverse dose-response association was observed between physical activity and colon cancer in 48 studies including 40,674 colon/colorectal cancer cases for both sexes. A dose-response effect of physical activity on colon cancer risk was especially observed, when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed as MET-hours per week. An observed inverse association with a dose-response relationship between physical activity and breast cancer was also identified in the majority of the 41 studies including 108,031 breast cancer cases. The dose-response relationship was in particular observed in case-control studies and supported by observations in cohort studies when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed by MET-hours per week. This association between physical activity and breast cancer risk is possibly dependent on age at exposure, age at diagnosis, menopausal status and other effect

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eren, Elif; Murphy, Megan; Goguen, Jon

    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 changesmore » 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.« less

  10. 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

  11. Water in the active site of ketosteroid isomerase.

    PubMed

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-08-09

    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 in the Y16S mutant and one in the Y16F and FFF mutants, with 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 (1)H 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 probable

  12. Normal Modes Expose Active Sites in Enzymes.

    PubMed

    Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

    2016-12-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.

  13. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  14. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

    PubMed Central

    Treuheit, Nicholas A.; Beach, Muneera A.; Komives, Elizabeth A.

    2011-01-01

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethylketone to the active site serine, as well as non-covalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1, however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-L-arginine-(3-methyl-1,5-pantanediyl) amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause the same reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or to exosite 1. PMID:21526769

  15. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

    PubMed

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

    2011-05-31

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  16. Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

    DOE PAGES

    Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping; ...

    2017-07-27

    Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

  17. Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping

    Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

  18. Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates.

    PubMed

    Devine, Erin L; Theobald, Douglas L; Oprian, Daniel D

    2016-08-30

    The visual pigment rhodopsin is a G protein-coupled receptor that covalently binds its retinal chromophore via a Schiff base linkage to an active-site Lys residue in the seventh transmembrane helix. Although this residue is strictly conserved among all type II retinylidene proteins, we found previously that the active-site Lys in bovine rhodopsin (Lys296) can be moved to three other locations (G90K, T94K, S186K) while retaining the ability to form a pigment with retinal and to activate transducin in a light-dependent manner [ Devine et al. ( 2013 ) Proc. Natl. Acad. Sci. USA 110 , 13351 - 13355 ]. Because the active-site Lys is not functionally constrained to be in helix seven, it is possible that it could relocate within the protein, most likely via an evolutionary intermediate with two active-site Lys. Therefore, in this study we characterized potential evolutionary intermediates with two Lys in the active site. Four mutant rhodopsins were prepared in which the original Lys296 was left untouched and a second Lys residue was substituted for G90K, T94K, S186K, or F293K. All four constructs covalently bind 11-cis-retinal, form a pigment, and activate transducin in a light-dependent manner. These results demonstrate that rhodopsin can tolerate a second Lys in the retinal binding pocket and suggest that an evolutionary intermediate with two Lys could allow migration of the Schiff base Lys to a position other than the observed, highly conserved location in the seventh TM helix. From sequence-based searches, we identified two groups of natural opsins, insect UV cones and neuropsins, that contain Lys residues at two positions in their active sites and also have intriguing spectral similarities to the mutant rhodopsins studied here.

  19. Psychophysiological responses to subliminal auditory suggestions for activation.

    PubMed

    Borgeat, F; Boissonneault, J; Chaloult, L; Elie, R

    1989-12-01

    The aim of this study was to characterize further the experimental procedure proposed by Borgeat and Goulet in 1983. Subliminal activation auditory suggestions were expected to increase heart rate and skin conductance in presence of a stressing task. 20 subjects were alternately exposed to activating or neutral (random numbers) 25-dB suggestions masked by a 40-dB white noise. Analyses of variance and covariance indicated an effect of the activating suggestions on heart rate but not on skin conductance. Partly confirming previous results, the data support the reliability of heart rate and the importance of a stressing factor to reveal the effects of preconscious or subliminal stimulations.

  20. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites.

    PubMed

    Chen, Baoyu; Chou, Hui-Ting; Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas; Rosen, Michael K

    2017-09-26

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

  1. Analysis of proton wires in the enzyme active site suggests a mechanism of c-di-GMP hydrolysis by the EAL domain phosphodiesterases.

    PubMed

    Grigorenko, Bella L; Knyazeva, Marina A; Nemukhin, Alexander V

    2016-11-01

    We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics.

    PubMed

    Bauler, Patricia; Huber, Gary; Leyh, Thomas; McCammon, J Andrew

    2010-05-06

    Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.

  3. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben

    2017-03-28

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by themore » SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.« less

  4. The N253F mutant structure of trehalose synthase from Deinococcus radiodurans reveals an open active-site topology.

    PubMed

    Chow, Sih Yao; Wang, Yung Lin; Hsieh, Yu Chiao; Lee, Guan Chiun; Liaw, Shwu Huey

    2017-11-01

    Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures of Deinococcus radiodurans TS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes.

  5. A suggested color scheme for reducing perception-related accidents on construction work sites.

    PubMed

    Yi, June-seong; Kim, Yong-woo; Kim, Ki-aeng; Koo, Bonsang

    2012-09-01

    Changes in workforce demographics have led to the need for more sophisticated approaches to addressing the safety requirements of the construction industry. Despite extensive research in other industry domains, the construction industry has been passive in exploring the impact of a color scheme; perception-related accidents have been effectively diminished by its implementation. The research demonstrated that the use of appropriate color schemes could improve the actions and psychology of workers on site, thereby increasing their perceptions of potentially dangerous situations. As a preliminary study, the objects selected by rigorous analysis on accident reports were workwear, safety net, gondola, scaffolding, and safety passage. The colors modified on site for temporary facilities were adopted from existing theoretical and empirical research that suggests the use of certain colors and their combinations to improve visibility and conspicuity while minimizing work fatigue. The color schemes were also tested and confirmed through two workshops with workers and managers currently involved in actual projects. The impacts of color schemes suggested in this paper are summarized as follows. First, the color schemes improve the conspicuity of facilities with other on site components, enabling workers to quickly discern and orient themselves in their work environment. Secondly, the color schemes have been selected to minimize the visual work fatigue and monotony that can potentially increase accidents. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. 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

  7. Studies and Suggestions on Prewriting Activities

    ERIC Educational Resources Information Center

    Zheng, Shigao; Dai, Weiping

    2012-01-01

    This paper studies and suggests the need for writing instruction by which students can experience writing as a creative process in exploring and communicating meaning. The prewriting activities generate ideas which can encourage a free flow of thoughts and help students discover both what they want to say and how to say it on paper. Through the…

  8. 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?

  9. Heterogeneous Electrocatalyst with Molecular Cobalt Ions Serving as the Center of Active Sites.

    PubMed

    Wang, Jiong; Ge, Xiaoming; Liu, Zhaolin; Thia, Larissa; Yan, Ya; Xiao, Wei; Wang, Xin

    2017-02-08

    Molecular Co 2+ ions were grafted onto doped graphene in a coordination environment, resulting in the formation of molecularly well-defined, highly active electrocatalytic sites at a heterogeneous interface for the oxygen evolution reaction (OER). The S dopants of graphene are suggested to be one of the binding sites and to be responsible for improving the intrinsic activity of the Co sites. The turnover frequency of such Co sites is greater than that of many Co-based nanostructures and IrO 2 catalysts. Through a series of carefully designed experiments, the pathway for the evolution of the Co cation-based molecular catalyst for the OER was further demonstrated on such a single Co-ion site for the first time. The Co 2+ ions were successively oxidized to Co 3+ and Co 4+ states prior to the OER. The sequential oxidation was coupled with the transfer of different numbers of protons/hydroxides and generated an active Co 4+ ═O fragment. A side-on hydroperoxo ligand of the Co 4+ site is proposed as a key intermediate for the formation of dioxygen.

  10. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  11. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

    PubMed

    Blaha-Nelson, David; Krüger, Dennis M; Szeler, Klaudia; Ben-David, Moshe; Kamerlin, Shina Caroline Lynn

    2017-01-25

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the k cat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.

  12. McIntosh active-region class similarities and suggestions for mergers

    NASA Technical Reports Server (NTRS)

    Bornmann, P. L.; Kalmbach, D.; Kulhanek, D.

    1994-01-01

    McIntosh active-region classifications reported during a five-year period were examined to determine similarities among the classes. Two methods were used extensively to determine these similarities. The number of transitions among classes were used to determine the most frequent transitions out of each class, and the alternative classes reported for the same region by different sites were used to establish which classes were neighboring classes. These transition frequencies and neighboring classes were used to identify classes that could be eliminated or merged with other classes. Class similarities were used to investigate the relative importance of several pairs of decisions that occur within a single McIntosh parameter. In particular, the redundancy of parameters in some classes was examined, and the class similarities were used to identify which of these parameters could be eliminated. Infrequently reported classes were also considered, and suggestions for mergers were made when similarities between classes could be identified.

  13. Dynamic asymmetry and the role of the conserved active-site thiol in rabbit muscle creatine kinase.

    PubMed

    Londergan, Casey H; Baskin, Rachel; Bischak, Connor G; Hoffman, Kevin W; Snead, David M; Reynoso, Christopher

    2015-01-13

    Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a

  14. Active site dynamics of ribonuclease.

    PubMed Central

    Brünger, A T; Brooks, C L; Karplus, M

    1985-01-01

    The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state. Images PMID:3866234

  15. 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

  16. 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

  17. Interactive flare sites within an active region complex

    NASA Technical Reports Server (NTRS)

    Poletto, G.; Gary, G. A.; Machado, M. E.

    1993-01-01

    We examine here a set of images of an active region complex, acquired on June 24-25, 1980, by the Hard X-ray Imaging Spectrometer on SMM, with the purpose of establishing whether there was any interplay between the frequent activity observed at different sites in the activity center and, in such a case, how the interaction was established. By analyzing both quiet and active orbits we show that, as a rule, activity originating in one region triggers the other region's activity. However, we find little unambiguous evidence for the presence of large-scale interconnecting loops. A comparison of X-ray images with magnetic field observations suggested that we interpret the active region behavior in terms of the interaction between different loop systems, in a scenario quite analogous to the interacting bipole representation of individual flares. We conclude that active region interplay provides an easily observable case to study the time-dependent topology and the mechanisms for the spreading of activity in transient events over all energy scales.

  18. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Carra,J.; McHugh, C.; Mulligan, S.

    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 tomore » a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.« less

  19. Psychophysiological changes following auditory subliminal suggestions for activation and deactivation.

    PubMed

    Borgeat, F; Goulet, J

    1983-06-01

    This study was to measure eventual psychophysiological changes resulting from auditory subliminal activation or deactivation suggestions. 18 subjects were alternately exposed to a control situation and to 25-dB activating and deactivating suggestions masked by a 40-dB white noise. Physiological measures (EMG, heart rate, skin-conductance levels and responses, and skin temperature) were recorded while subjects listened passively to the suggestions, during a stressing task that followed and after that task. Multivariate analysis of variance showed a significant effect of the activation subliminal suggestions during and following the stressing task. This result is discussed as indicating effects of consciously unrecognized perceptions on psychophysiological responses.

  20. What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

    PubMed

    Zhang, Ni; Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

    2017-06-22

    Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. ©Ni Zhang, Shelly Campo, Jingzhen Yang, Petya Eckler, Linda Snetselaar, Kathleen Janz, Emily Leary. Originally published in the Journal of Medical

  1. What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

    PubMed Central

    Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

    2017-01-01

    Background Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. Objective This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. Methods A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. Results The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. Conclusions This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. PMID:28642215

  2. 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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  4. Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

    PubMed Central

    2015-01-01

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

  5. Nest site selection by Kentish plover suggests a trade-off between nest-crypsis and predator detection strategies.

    PubMed

    Gómez-Serrano, Miguel Ángel; López-López, Pascual

    2014-01-01

    Predation is one of the main causes of adult mortality and breeding failure for ground-nesting birds. Micro-habitat structure around nests plays a critical role in minimizing predation risk. Plovers nest in sites with little vegetation cover to maximize the incubating adult visibility, but many studies suggest a trade-off between nest-crypsis and predator detection strategies. However, this trade-off has not been explored in detail because methods used so far do not allow estimating the visibility with regards to critical factors such as slope or plant permeability to vision. Here, we tested the hypothesis that Kentish plovers select exposed sites according to a predator detection strategy, and the hypothesis that more concealed nests survive longer according to a crypsis strategy. To this end, we obtained an accurate estimation of the incubating adult's field of vision through a custom built inverted periscope. Our results showed that plovers selected nest sites with higher visibility than control points randomly selected with regards to humans and dogs, although nests located in sites with higher vegetation cover survived longer. In addition, the flushing distance (i.e., the distance at which incubating adults leave the nest when they detect a potential predator) decreased with vegetation cover. Consequently, the advantages of concealing the nest were limited by the ability to detect predators, thus indirectly supporting the existence of the trade-off between crypsis and predator detection. Finally, human disturbance also constrained nest choice, forcing plovers to move to inland sites that were less suitable because of higher vegetation cover, and modulated flushing behavior, since plovers that were habituated to humans left their nests closer to potential predators. This constraint on the width of suitable breeding habitat is particularly relevant for the conservation of Kentish Plover in sand beaches, especially under the current context of coastal regression

  6. Nest Site Selection by Kentish Plover Suggests a Trade-Off between Nest-Crypsis and Predator Detection Strategies

    PubMed Central

    Gómez-Serrano, Miguel Ángel; López-López, Pascual

    2014-01-01

    Predation is one of the main causes of adult mortality and breeding failure for ground-nesting birds. Micro-habitat structure around nests plays a critical role in minimizing predation risk. Plovers nest in sites with little vegetation cover to maximize the incubating adult visibility, but many studies suggest a trade-off between nest-crypsis and predator detection strategies. However, this trade-off has not been explored in detail because methods used so far do not allow estimating the visibility with regards to critical factors such as slope or plant permeability to vision. Here, we tested the hypothesis that Kentish plovers select exposed sites according to a predator detection strategy, and the hypothesis that more concealed nests survive longer according to a crypsis strategy. To this end, we obtained an accurate estimation of the incubating adult's field of vision through a custom built inverted periscope. Our results showed that plovers selected nest sites with higher visibility than control points randomly selected with regards to humans and dogs, although nests located in sites with higher vegetation cover survived longer. In addition, the flushing distance (i.e., the distance at which incubating adults leave the nest when they detect a potential predator) decreased with vegetation cover. Consequently, the advantages of concealing the nest were limited by the ability to detect predators, thus indirectly supporting the existence of the trade-off between crypsis and predator detection. Finally, human disturbance also constrained nest choice, forcing plovers to move to inland sites that were less suitable because of higher vegetation cover, and modulated flushing behavior, since plovers that were habituated to humans left their nests closer to potential predators. This constraint on the width of suitable breeding habitat is particularly relevant for the conservation of Kentish Plover in sand beaches, especially under the current context of coastal regression

  7. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 materialmore » 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.« less

  8. Divergent and convergent evolution in metastases suggest treatment strategies based on specific metastatic sites

    PubMed Central

    Cunningham, Jessica J.; Brown, Joel S.; Vincent, Thomas L.

    2015-01-01

    Background and objective: Systemic therapy for metastatic cancer is currently determined exclusively by the site of tumor origin. Yet, there is increasing evidence that the molecular characteristics of metastases significantly differ from the primary tumor. We define the evolutionary dynamics of metastases that govern this molecular divergence and examine their potential contribution to variations in response to targeted therapies. Methodology: Darwinian interactions of transformed cells with the tissue microenvironments at primary and metastatic sites are analyzed using evolutionary game theory. Computational models simulate responses to targeted therapies in different organs within the same patient. Results: Tumor cells, although maximally fit at their primary site, typically have lower fitness on the adaptive landscapes offered by the metastatic sites due to organ-specific variations in mesenchymal properties and signaling pathways. Clinically evident metastases usually exhibit time-dependent divergence from the phenotypic mean of the primary population as the tumor cells evolve and adapt to their new circumstances. In contrast, tumors from different primary sites evolving on identical metastatic adaptive landscapes exhibit phenotypic convergence. Thus, metastases in the liver from different primary tumors and even in different hosts will evolve toward similar adaptive phenotypes. The combination of evolutionary divergence from the primary cancer phenotype and convergence towards similar adaptive strategies in the same tissue cause significant variations in treatment responses particularly for highly targeted therapies. Conclusion and implications: The results suggest that optimal therapies for disseminated cancer must take into account the site(s) of metastatic growth as well as the primary organ. PMID:25794501

  9. Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form.

    PubMed

    Lee, Yie-Vern; Choi, Sy Bing; Wahab, Habibah A; Choong, Yee Siew

    2017-09-25

    Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C 2 -C 3 bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.

  10. Carbinolamine Formation and Dehydration in a DNA Repair Enzyme Active Site

    PubMed Central

    Dodson, M. L.; Walker, Ross C.; Lloyd, R. Stephen

    2012-01-01

    In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics–molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water–independent enzyme–catalyzed reaction had a bias–corrected Jarzynski–average barrier height of approximately for the carbinolamine formation reaction and ) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately in the forward (formation) reaction and for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water–independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N–terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site

  11. Evidence for histidine in the active sites of ficin and stem-bromelain

    PubMed Central

    Husain, S. S.; Lowe, G.

    1968-01-01

    1. Ficin and stem-bromelain are irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. Evidence is presented that establishes that a histidine residue is within a 5Å locus of the active-site cysteine residue in both enzymes. The histidine residue in both enzymes is alkylated at N-1 by dibromoacetone. It is suggested that, as with papain, the thiol and imidazole groups act in concert in the hydrolysis of substrates by these enzymes. 2. The inhibition of thiol-subtilisin with 1,3-dibromoacetone is shown to be due to the alkylation of a cysteine residue only. PMID:5722692

  12. 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

  13. Discriminative structural approaches for enzyme active-site prediction.

    PubMed

    Kato, Tsuyoshi; Nagano, Nozomi

    2011-02-15

    Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

  14. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    DOE PAGES

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open'more » structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. In conclusion, as a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

  15. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

    PubMed

    Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  16. Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

    PubMed

    Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

    2016-03-01

    An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures.

  17. Ionizable side chains at catalytic active sites of enzymes.

    PubMed

    Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

    2012-05-01

    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 1,072 Å(3). 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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.

    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 uponmore » 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.« less

  20. Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

    PubMed

    Adamczyk, Katrin; Simpson, Niall; Greetham, Gregory M; Gumiero, Andrea; Walsh, Martin A; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

    2015-01-01

    Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H 2 O and D 2 O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump-probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.

  1. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Phillips, J.; Warby, C; Whitby, F

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connectedmore » by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.« less

  2. 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.

  3. Quantum mechanical design of enzyme active sites.

    PubMed

    Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

    2008-02-01

    The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

  4. Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

    PubMed

    Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

    2017-05-01

    RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.

  5. A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity.

    PubMed

    Tilvawala, Ronak; Cammarata, Michael; Adediran, S A; Brodbelt, Jennifer S; Pratt, R F

    2015-12-22

    O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

  6. Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

    NASA Technical Reports Server (NTRS)

    Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

    2000-01-01

    Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

  7. A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

    PubMed

    Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

    2016-10-04

    Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

  8. High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

    PubMed

    Romi, Erez; Baran, Nava; Gantman, Marina; Shmoish, Michael; Min, Bosun; Collins, Kathleen; Manor, Haim

    2007-05-22

    Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

  9. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. © 2010 The Authors Journal compilation © 2010 FEBS.

  10. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  11. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

    DOE PAGES

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

    2014-12-31

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

  12. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

  13. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  14. Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

    PubMed

    Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

    1999-03-01

    The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

  15. Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

    PubMed

    Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

    2016-11-15

    The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

  16. Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

    PubMed

    Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

    2018-02-02

    Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. 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

  18. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

    PubMed Central

    Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-01-01

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  19. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  20. Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease.

    PubMed

    Ragland, Debra A; Nalivaika, Ellen A; Nalam, Madhavi N L; Prachanronarong, Kristina L; Cao, Hong; Bandaranayake, Rajintha M; Cai, Yufeng; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-08-27

    HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

  1. Parkin-phosphoubiquitin complex reveals a cryptic ubiquitin binding site required for RBR ligase activity

    PubMed Central

    Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

    2017-01-01

    RING-BETWEENRING-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR is Parkin, mutations in which lead to early onset hereditary Parkinsonism. Parkin and other RBRs share a catalytic RBR module, but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during activation of Parkin. However, current data on active RBRs are in the absence of regulatory domains. Therefore, how individual RBRs are activated, and whether they share a common mechanism remains unclear. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces a movement in the IBR domain to reveal a cryptic ubiquitin binding site. Mutation of this site negatively impacts on Parkin’s activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, suggesting a role for interdomain association in RBR ligase mechanism. PMID:28414322

  2. The role of an active site Mg(2+) in HDV ribozyme self-cleavage: insights from QM/MM calculations.

    PubMed

    Mlýnský, Vojtěch; Walter, Nils G; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

    2015-01-07

    The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H(+) in the active site, which acts as the general acid, and a partially hydrated Mg(2+) ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with a distinct position and coordination of the catalytically important active site Mg(2+) ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal mol(-1), indicating that the specific position of the Mg(2+) ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2'-OH) nucleophile and the nucleophilic attack of the resulting U-1(2'-O(-)) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimate the pKa of the U-1(2'-OH) group to range from 8.8 to 11.2, suggesting that it is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg(2+) ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg(2+) ion, facilitate deprotonation and activation of the 2'-OH nucleophile.

  3. Direct instrumental identification of catalytically active surface sites

    NASA Astrophysics Data System (ADS)

    Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

    2017-09-01

    The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

  4. The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

    PubMed Central

    Just, Victoria J.; Burrell, Matthew R.; Bowater, Laura; McRobbie, Iain; Stevenson, Clare E. M.; Lawson, David M.; Bornemann, Stephen

    2007-01-01

    Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural. PMID:17680775

  5. Uncovering the determinants of a highly perturbed tyrosine pKa in the active site of ketosteroid isomerase.

    PubMed

    Schwans, Jason P; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

    2013-11-05

    Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, (13)C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural

  6. Uncovering the Determinants of a Highly Perturbed Tyrosine pKa in the Active Site of Ketosteroid Isomerase†

    PubMed Central

    Schwans, Jason P.; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

    2013-01-01

    Within the idiosyncratic enzyme active site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near to charged groups or within hydrophobic pockets, Tyr57 of a P. putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semi-synthesis, 13C NMR spectroscopy, absorbance spectroscopy, and x-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from introduction of nearby anionic groups arise from accompanying active site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen bond donors, two Tyr side chains and a water molecule, positioned by other side chains and by a water-mediated hydrogen bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions, rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can

  7. Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

    PubMed Central

    Dahms, Sven O.; Arciniega, Marcelino; Steinmetzer, Torsten; Huber, Robert; Than, Manuel E.

    2016-01-01

    Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8–2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the “off state,” incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active “on state” seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin. PMID:27647913

  8. Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

    PubMed

    Hwang, D C; Kim, D H; Lee, J S; Kang, B H; Han, J; Kim, W; Song, B D; Choi, K Y

    2000-10-31

    Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

  9. Redefinition of rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues.

    PubMed

    Kannappan, Babu; Gready, Jill E

    2008-11-12

    Crystallographic, mutagenesis, kinetic, and computational studies on Rubisco over three decades have revealed much about its catalytic mechanism and the role played by several active-site residues. However, key questions remain unanswered. Specific details of the carboxylase and oxygenase mechanisms, required to underpin the rational re-engineering of Rubisco, are still speculative. Here we address critical gaps in knowledge with a definitive comprehensive computational investigation of the mechanism of carboxylase activity at the Rubisco active site. Density functional theory calculations (B3LYP/6-31G(d,p)) were performed on active-site fragment models of a size up to 77 atoms, not previously possible computationally. All amino acid residues suspected to play roles in the acid-base chemistry in the multistep reaction, and interacting directly with the central Mg (2+) atom and the reactive moiety of substrate and intermediates, were included. The results provide a firm basis for us to propose a novel mechanism for the entire sequence of reactions in the carboxylase catalysis and to define precise roles for the active-site residues, singly and in concert. In this mechanism, the carbamylated LYS201 plays a more limited role than previously proposed but is crucial for initiating the reaction by acting as a base in the enolization. We suggest a wider role for HIS294, with involvement in the carboxylation, hydration, and C2-C3 bond-scission steps, consistent with the suggestion of Harpel et al. (1998) but contrary to the consensus view of Cleland et al. (1998). In contrast to the common assumption that the water molecule for the hydration step comes from within the active site, we propose that the Mg-coordinated water is not dissociated at the start of the gas-addition reaction but rather remains coordinated and is used for the hydration of the C3 carbon atom. New roles are also proposed for LYS175, GLU204, and HIS294. The mechanism suggests roles in the gas

  10. Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions.

    PubMed

    Ebert, Maximilian C C J C; Morley, Krista L; Volpato, Jordan P; Schmitzer, Andreea R; Pelletier, Joelle N

    2015-04-01

    Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation. © 2014 The Protein Society.

  11. Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

    PubMed

    Gray, K A; Dutton, P L; Daldal, F

    1994-01-25

    Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

  12. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  13. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  14. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  15. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  16. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  17. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

    PubMed

    Kumar, Raj; Calhoun, William J

    2008-12-01

    Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and

  18. Active-site monovalent cations revealed in a 1.55-Å-resolution hammerhead ribozyme structure.

    PubMed

    Anderson, Michael; Schultz, Eric P; Martick, Monika; Scott, William G

    2013-10-23

    We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under 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 with 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 that of 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong

    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 influenzamore » 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.« less

  20. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

    PubMed

    de San Martin, Javier Zorrilla; Jalil, Abdelali; Trigo, Federico F

    2015-12-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca(2+) photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl(-)](i), autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30-150 GABA(A) channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na(v)-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. © 2015 Zorrilla de San Martin et al.

  1. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  2. Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii.

    PubMed

    Pratap, Shivendra; Katiki, Madhusudhanarao; Gill, Preet; Kumar, Pravindra; Golemi-Kotra, Dasantila

    2016-01-01

    Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

    PubMed

    Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

    2016-09-12

    CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.

  4. Activities suggestion based on emotions in AAL environments.

    PubMed

    Costa, Angelo; Rincon, Jaime Andres; Carrascosa, Carlos; Novais, Paulo; Julian, Vicente

    2018-03-01

    The elderly population is increasing and the response of the society was to provide them with services directed to them to cope with their needs. One of the oldest solutions is the retirement home, providing housing and permanent assistance for the elderly. Furthermore, most of the retirement homes are inhabited by multiple elderly people, thus creating a community of people who are somewhat related in age and medical issues. The ambient assisted living (AAL) area tries to solve some of the elderly issues by producing technological products, some of them dedicated to elderly homes. One of the identified problem is that elderly people are sometimes discontent about the activities that consume most of their day promoted by the retirement home social workers. The work presented in this paper attempts to improve how these activities are scheduled taking into account the elderlies' emotional response to these activities. The aim is to maximize the group happiness by promoting the activities the group likes, minding if they are bored due to activities repetition. In this sense, this paper presents an extension of the Cognitive Life Assistant platform incorporating a social emotional model. The proposed system has been modelled as a free time activity manager which is in charge of suggesting activities to the social workers. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

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

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  8. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  9. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  10. Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

    PubMed

    Asgeirsson, Bjarni; Adalbjörnsson, Björn Vidar; Gylfason, Gudjón Andri

    2007-06-01

    Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

  11. Evolution of a designed retro-aldolase leads to complete active site remodeling

    PubMed Central

    Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

    2013-01-01

    Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

  12. A gratuitous β-Lactamase inducer uncovers hidden active site dynamics of the Staphylococcus aureus BlaR1 sensor domain.

    PubMed

    Frederick, Thomas E; Peng, Jeffrey W

    2018-01-01

    Increasing evidence shows that active sites of proteins have non-trivial conformational dynamics. These dynamics include active site residues sampling different local conformations that allow for multiple, and possibly novel, inhibitor binding poses. Yet, active site dynamics garner only marginal attention in most inhibitor design efforts and exert little influence on synthesis strategies. This is partly because synthesis requires a level of atomic structural detail that is frequently missing in current characterizations of conformational dynamics. In particular, while the identity of the mobile protein residues may be clear, the specific conformations they sample remain obscure. Here, we show how an appropriate choice of ligand can significantly sharpen our abilities to describe the interconverting binding poses (conformations) of protein active sites. Specifically, we show how 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) exposes otherwise hidden dynamics of a protein active site that binds β-lactam antibiotics. When CBAP acylates (binds) the active site serine of the β-lactam sensor domain of BlaR1 (BlaRS), it shifts the time scale of the active site dynamics to the slow exchange regime. Slow exchange enables direct characterization of inter-converting protein and bound ligand conformations using NMR methods. These methods include chemical shift analysis, 2-d exchange spectroscopy, off-resonance ROESY of the bound ligand, and reduced spectral density mapping. The active site architecture of BlaRS is shared by many β-lactamases of therapeutic interest, suggesting CBAP could expose functional motions in other β-lactam binding proteins. More broadly, CBAP highlights the utility of identifying chemical probes common to structurally homologous proteins to better expose functional motions of active sites.

  13. Computer simulation of the active site of human serum cholinesterase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positivemore » electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.« less

  14. Experimental warming differentially affects microbial structure and activity in two contrasted moisture sites in a Sphagnum-dominated peatland.

    PubMed

    Delarue, Frédéric; Buttler, Alexandre; Bragazza, Luca; Grasset, Laurent; Jassey, Vincent E J; Gogo, Sébastien; Laggoun-Défarge, Fatima

    2015-04-01

    Several studies on the impact of climate warming have indicated that peat decomposition/mineralization will be enhanced. Most of these studies deal with the impact of experimental warming during summer when prevalent abiotic conditions are favorable to decomposition. Here, we investigated the effect of experimental air warming by open-top chambers (OTCs) on water-extractable organic matter (WEOM), microbial biomasses and enzymatic activities in two contrasted moisture sites named Bog and Fen sites, the latter considered as the wetter ones. While no or few changes in peat temperature and water content appeared under the overall effect of OTCs, we observed that air warming smoothed water content differences and led to a decrease in mean peat temperature at the warmed Bog sites. This thermal discrepancy between the two sites led to contrasting changes in microbial structure and activities: a rise in hydrolytic activity at the warmed Bog sites and a relative enhancement of bacterial biomass at the warmed Fen sites. These features were not associated with any change in WEOM properties namely carbon and sugar contents and aromaticity, suggesting that air warming did not trigger any shift in OM decomposition. Using various tools, we show that the use of single indicators of OM decomposition can lead to fallacious conclusions. Lastly, these patterns may change seasonally as a consequence of complex interactions between groundwater level and air warming, suggesting the need to improve our knowledge using a high time-resolution approach. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins.

    PubMed

    Chakravorty, David; Trusov, Yuri; Botella, José Ramón

    2012-03-01

    Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins.

  16. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  17. Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements

    PubMed Central

    Bridgewater, Laura C.; Walker, Marlan D.; Miller, Gwen C.; Ellison, Trevor A.; Holsinger, L. Daniel; Potter, Jennifer L.; Jackson, Todd L.; Chen, Reuben K.; Winkel, Vicki L.; Zhang, Zhaoping; McKinney, Sandra; de Crombrugghe, Benoit

    2003-01-01

    Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5′ region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled. PMID:12595563

  18. 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

  19. 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.

  20. Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes.

    PubMed

    Zastrow, Melissa L; Pecoraro, Vincent L

    2013-04-17

    While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

  1. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology.

    PubMed

    Rabey, Karyne N; Green, David J; Taylor, Andrea B; Begun, David R; Richmond, Brian G; McFarlin, Shannon C

    2015-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual's past behaviour. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology

    PubMed Central

    Rabey, Karyne N.; Green, David J.; Taylor, Andrea B.; Begun, David R.; Richmond, Brian G.; McFarlin, Shannon C.

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 ofmore » vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.« less

  4. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarilymore » at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.« less

  5. HTO and OBT activity concentrations in soil at the historical atmospheric HT release site (Chalk River Laboratories).

    PubMed

    Kim, S B; Bredlaw, M; Korolevych, V Y

    2012-01-01

    Tritium is routinely released by the Chalk River Laboratories (CRL) nuclear facilities. Three International HT release experiments have been conducted at the CRL site in the past. The site has not been disturbed since the last historical atmospheric testing in 1994 and presents an opportunity to assess the retention of tritium in soil. This study is devoted to the measurement of HTO and OBT activity concentration profiles in the subsurface 25 cm of soil. In terms of soil HTO, there is no evidence from the past HT release experiments that HTO was retained. The HTO activity concentration in the soil pore water appears similar to concentrations found in background areas in Ontario. In contrast, OBT activity concentrations in soil at the same site were significantly higher than HTO activity concentrations in soil. Elevated OBT appears to reside in the top layer of the soil (0-5 cm). In addition, OBT activity concentrations in the top soil layer did not fluctuate much with season, again, quite in contrast with soil HTO. This result suggests that OBT activity concentrations retained the signature of the historical tritium releases. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  6. Unveiling the water-associated conformational mobility in the active site of ascorbate peroxidase.

    PubMed

    Chao, Wei-Chih; Lin, Li-Ju; Lu, Jyh-Feng; Wang, Jinn-Shyan; Lin, Tzu-Chieh; Chen, Yi-Han; Chen, Yi-Ting; Yang, Hsiao-Ching; Chou, Pi-Tai

    2018-03-01

    We carried out comprehensive spectroscopic studies of wild type and mutants of ascorbate peroxidase (APX) to gain understanding of the conformational mobility of the active site. In this approach, three unnatural tryptophans were applied to replace the distal tryptophan (W41) in an aim to probe polarity/water environment near the edge of the heme-containing active site. 7-azatryptophan ((7-aza)Trp) is sensitive to environment polarity, while 2,7-azatryptophan ((2,7-aza)Trp) and 2,6-diazatryptophan ((2,6-aza)Trp) undergo excited-state water-catalyzed double and triple proton transfer, respectively, and are sensitive to the water network. The combination of their absorption, emission bands and the associated relaxation dynamics of these fluorescence probes, together with the Soret-band difference absorption and resonance Raman spectroscopy, lead us to unveil the water associated conformational mobility in the active site of APX. The results are suggestive of the existence of equilibrium between two different environments surrounding W41 in APX, i.e., the water-rich and water-scant forms with distinct fluorescence relaxation. Our results thus demonstrate for the first time the power of integrating multiple sensors (7-aza)Trp, (2,7-aza)Trp and (2,6-aza)Trp in probing the water environment of a specifically targeted Trp in proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

    PubMed Central

    Sigala, Paul A.; Kraut, Daniel A.; Caaveiro, Jose M. M.; Pybus, Brandon; Ruben, Eliza A.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel

    2009-01-01

    Enzymes are classically proposed to accelerate reactions by binding substrates within active site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1 – 1 Å scale of most substrate rearrangements. The flexibility of active site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together high resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition state analog binding measurements to test the distance scale on which non-covalent forces can constrain side chain and ligand relaxation or translation along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side chain reorientation and prevent hydrogen bond shortening by 0.1 Å or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function. PMID:18808119

  8. 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

  9. Effects of hexamethonium, phenothiazines, propranolol and ephedrine on acetylcholinesterase carbamylation by physostigmine, aldicarb and carbaryl: interaction between the active site and the functionally distinct peripheral sites in acetylcholinesterase.

    PubMed

    Singh, A K; Spassova, D

    1998-01-01

    Physostigmine, aldicarb and carbaryl were potent inhibitors of acetylcholinesterase (AChE). The physostigmine-inhibited AChE fluoresced at 300 nm excitation and 500 nm emission wavelengths, but the aldicarb and carbaryl inhibited enzyme did not. This suggests that the carbamylated active center is not the fluorescing site in AChE. The fluorescence intensity of physostigmine-inhibited AChE decreased with increasing the substrate (acetylthiocholine) concentration, thus indicating that physostigmine binding to the active site is essential for the development of fluorescence. Thus, the physostigmine-inhibited AChE fluoresces due to the binding of trimethylpyrrolo[2,3-b]indol (TMPI) moiety, formed by the hydrolysis of physostigmine, to a peripheral site in AChE. The fluorescence intensity of the physostigmine-inhibited enzyme decreased when the inhibited-enzyme was dialyzed for either 30 min that poorly reactivated the enzyme or 180 min that fully reactivated the enzyme. This suggests that dialysis dissociates the AChE-TMPI complex much faster than it reactivates the carbamylated AChE. Ephedrine, propranolol and phenothiazines including trifluoparazine (TPZ) caused non-competitive inhibition, while hexamethonium caused an uncompetitive inhibition of AChE activity. TPZ, upon binding with AChE, formed a fluorescent TPZ-enzyme complex. The fluorescence intensity of TPZ-AChE complex was effectively decreased by ephedrine, but not by propranolol or hexamethonium. This indicates that TPZ and ephedrine bind to the same site in AChE which is different from the site/or sites to which propranolol or hexamethonium bind. Hexamethonium protected AChE from inhibition by carbamates and decreased the fluorescence intensity of the physostigmine-inhibited AChE. Phenothiazines and ephedrine did not modulate the enzyme inhibition or the fluorescence intensity of the physostigmine-inhibited AChE. Propranolol and TPZ potentiated the enzyme inhibition and increased the fluorescence intensity

  10. All the catalytic active sites of MoS 2 for hydrogen evolution

    DOE PAGES

    Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

    2016-11-29

    MoS 2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS 2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated tomore » be 7.5 s –1 (65–75 mV/dec), 3.2 s –1 (65–85 mV/dec), and 0.1 s –1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS 2 is higher than that in low crystalline quality MoS 2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

  11. Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity.

    PubMed

    Chen, Zhi-Wei; Datta, Saumen; Dubois, Jennifer L; Klinman, Judith P; Mathews, F Scott

    2010-08-31

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  12. A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

    PubMed

    Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2017-11-01

    The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthew, E.; Parfitt, A.G.; Sugden, D.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects ofmore » diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.« less

  14. Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

    PubMed Central

    Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

    2014-01-01

    Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

  15. Chapter 12: Daily Patterns of Marbled Murrelet Activity at Inland Sites

    Treesearch

    Nancy L. Naslund; Brian P. O’Donnell

    1995-01-01

    Patterns in the daily activity of Marbled Murrelets (Brachyramphus marmoratus) at inland sites has been studied throughout their range from California to Alaska. Murrelets are most active at inland sites around dawn, and to a lesser degree, at dusk. Throughout their range, peak levels of activity (detections) occur in the hour around dawn, but...

  16. Chemosynthetic microbial activity at Mid-Atlantic Ridge hydrothermal vent sites

    NASA Astrophysics Data System (ADS)

    Wirsen, Carl O.; Jannasch, Holger W.; Molyneaux, Stephen J.

    1993-06-01

    Chemosynthetic production of microbial biomass, determined by 14CO2 fixation and enzymatic (RuBisCo) activity, at the Mid-Atlantic Ridge (MAR) 23° and 26°N vent sites was found in various niches: warm water emissions, loosely rock-attached flocculent material, dense morphologically diverse bacterial mats covering the surfaces of polymetal sulfide deposits, and filamentous microbes on the carapaces of shrimp (Rimicaris exoculata). The bacterial mats on polymetal sulfide surfaces contained unicellular and filamentous bacteria which appeared to use as their chemolithotrophic electron or energy source either dissolved reduced minerals from vent emissions, mainly sulfur compounds, or solid metal sulfide deposits, mainly pyrite. Moderately thermophilic Chemosynthetic activity was observed in carbon dioxide fixation experiments and in enrichments, but no thermophilic aerobic sulfur oxidizers could be isolated. Both obligate and facultative chemoautotrophs growing at mesophilic temperatures were isolated from all chemosynthetically active surface scrapings. The obligate autotrophs could oxidize sterilized MAR natural sulfide deposits as well as technical pyrite at near neutral pH, in addition to dissolved reduced sulfur compounds. While the grazing by shrimp on the surface mats of MAR metal sulfide deposits was observed and deemed important, the animals' primary occurrence in dense swarms near vent emissions suggests that they were feeding at these sites, where conditions for Chemosynthetic growth of their filamentous microbial epiflora were optimal. The data show that the transformation of geothermal energy at the massive polymetal sulfide deposits of the MAR is based on the lithoautotrophic oxidation of soluble sulfides and pyrites into microbial biomass.

  17. Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

    PubMed Central

    O'Rourke, Patrick E. F.; Eadsforth, Thomas C.; Fyfe, Paul K.; Shepherd, Sharon M.; Hunter, William N.

    2011-01-01

    4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism

  18. Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

    PubMed Central

    2015-01-01

    The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

  19. Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes.

    PubMed

    Endrizzi, James A; Beernink, Peter T

    2017-11-01

    A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations. © 2017 The Protein Society.

  20. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  1. 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

  2. Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

    PubMed

    Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

    2014-10-01

    Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

  3. Active-site solvent replenishment observed during human carbonic anhydrase II catalysis.

    PubMed

    Kim, Jin Kyun; Lomelino, Carrie L; Avvaru, Balendu Sankara; Mahon, Brian P; McKenna, Robert; Park, SangYoun; Kim, Chae Un

    2018-01-01

    Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO 2 /HCO 3 - . Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO 2 pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (W I ') is observed next to the previously observed intermediate water W I , and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH - on CO 2 . This mechanism explains how the zinc-bound water (W Zn ) and W1 are replenished, which are directly responsible for the reconnection of the His64-mediated proton-transfer water network. This study provides the first 'physical' glimpse of how a water reservoir flows into the hCA II active site during its catalytic activity.

  4. Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes.

    PubMed

    Geeraerts, Zachary; Celis, Arianna I; Mayfield, Jeffery A; Lorenz, Megan; Rodgers, Kenton R; DuBois, Jennifer L; Lukat-Rodgers, Gudrun S

    2018-03-06

    O 2 -evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO 2 - ) to O 2 and Cl - . Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO 2 - , albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10 -9 M, the K D for the KpCld-CN - complex is 2 orders of magnitude smaller than that of DaCld-CN - and indicates an affinity for CN - that is greater than that of most heme proteins. The difference in CN - affinity between Kp- and DaClds is predominantly due to differences in k off . The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p K a of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 396 cm -1 ) and KpCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 356 cm -1 ) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.

  5. Vaccines and Internet: characteristics of the vaccine safety net web sites and suggested improvements.

    PubMed

    Martínez-Mora, Marta; Alvarez-Pasquín, María José; Rodríguez-Salvanés, Francisco

    2008-12-09

    The Internet contains a large amount of useful information on many subjects, but also information of doubtful quality. To help identify Web sites on vaccine safety that fulfil good practice, the Global Advisory Committee on Vaccine Safety of the World Health Organization (WHO) has published criteria to which sites should adhere and a listing of Web sites that fulfil them. There are no studies describing the common attributes of these sites. To examine the attributes, design characteristics and resources of Web sites belonging to the Vaccine Safety Net (VSN) of the WHO. A cross-sectional, descriptive observational study using an evaluation questionnaire was carried out applied to the VSN web sites listed in March-April 2007. Twenty-six Web sites accredited by the VSN by April 2007 were analysed. With respect to design and quality, all sites contained information about the site manager. Postal and Email addresses were available for 84.6% of the sites. About privacy and personal data processing, 73.1% of sites specified the data protection procedure used and stated that data were not sold or passed third parties. The most-used language was English (76.9%). 96.3% of sites had links to other pro-vaccination sites and 19.2% provided the addresses of vaccination centres. 63.6% of webs were aimed at general public and health care workers but there was no clear separation of documents or different entry routes. With respect to information on vaccine safety, 84.6% of sites had information on adverse effects. In the general information section, 92.3% of sites had a new developments section. Some sites had multiple sources of financing and in 57% of sites, the financing was public. The most-important plus factors found were the transparency of financing, the lack of links to the pharmaceutical industry, the transparency of site management and responsibility and the proven scientific quality and constant updating of contents.

  6. The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.

    PubMed

    Goring, Mark E; Leibovitch, Matthew; Gea-Mallorqui, Ester; Karls, Shawn; Richard, Francis; Hanic-Joyce, Pamela J; Joyce, Paul B M

    2013-10-01

    We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures. © 2013.

  7. Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites.

    PubMed

    Ribeiro, António J M; Holliday, Gemma L; Furnham, Nicholas; Tyzack, Jonathan D; Ferris, Katherine; Thornton, Janet M

    2018-01-04

    M-CSA (Mechanism and Catalytic Site Atlas) is a database of enzyme active sites and reaction mechanisms that can be accessed at www.ebi.ac.uk/thornton-srv/m-csa. Our objectives with M-CSA are to provide an open data resource for the community to browse known enzyme reaction mechanisms and catalytic sites, and to use the dataset to understand enzyme function and evolution. M-CSA results from the merging of two existing databases, MACiE (Mechanism, Annotation and Classification in Enzymes), a database of enzyme mechanisms, and CSA (Catalytic Site Atlas), a database of catalytic sites of enzymes. We are releasing M-CSA as a new website and underlying database architecture. At the moment, M-CSA contains 961 entries, 423 of these with detailed mechanism information, and 538 with information on the catalytic site residues only. In total, these cover 81% (195/241) of third level EC numbers with a PDB structure, and 30% (840/2793) of fourth level EC numbers with a PDB structure, out of 6028 in total. By searching for close homologues, we are able to extend M-CSA coverage of PDB and UniProtKB to 51 993 structures and to over five million sequences, respectively, of which about 40% and 30% have a conserved active site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Polydactyly suggesting local husbandry of Pre-Columbian camelids: A case from Castillo de Huarmey archaeological site, northern coast of Peru.

    PubMed

    Tomczyk, Weronika; Giersz, Miłosz

    2017-03-01

    Three camelid metapodials with polydactyly (additional digits) were found at the Wari culture archaeological site (dated to the Middle Horizon) of Castillo de Huarmey. The anomalous bones were excavated among numerous remains, and presumably represent animals that were sacrificed within the principal mortuary mausoleum. The bones derive from at least two individuals. The etiology of the deformities remains unknown, but the most probable causes include low genetic diversity in the herd or unintended effect of selective breeding. The likelihood of impaired locomotion suggests birth and rearing within the site vicinity. The animals were juvenile, apparently killed around the age of sexual maturity, when they would have attained maximum body mass. Purposeful funerary proceedings with deformed animals suggest (at least) a locally developed camelid husbandry. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

    PubMed

    Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

    2018-05-03

    The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fang, Ti; Li, De-Feng; Zhou, Ning-Yi, E-mail: n.zhou@pentium.whiov.ac.cn

    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 isomerizationmore » 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

  11. Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures*

    PubMed Central

    Jain, Kanishk; Warmack, Rebeccah A.; Stavropoulos, Peter

    2016-01-01

    In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors. PMID:27387499

  12. Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures.

    PubMed

    Jain, Kanishk; Warmack, Rebeccah A; Debler, Erik W; Hadjikyriacou, Andrea; Stavropoulos, Peter; Clarke, Steven G

    2016-08-26

    In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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.

  14. SABER: A computational method for identifying active sites for new reactions

    PubMed Central

    Nosrati, Geoffrey R; Houk, K N

    2012-01-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were l-Ala d/l-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

  15. SABER: a computational method for identifying active sites for new reactions.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. Copyright © 2012 The Protein Society.

  16. Active sites of ligand-protected Au{sub 25} nanoparticle catalysts for CO{sub 2} electroreduction to CO

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alfonso, Dominic R., E-mail: alfonso@netl.doe.gov; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO{sub 2}) into CO at atomically precise negatively charged Au{sub 25}{sup −} nanoclusters. The studies showed CO{sub 2} 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 CO{sub 2} 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 CO{sub 2} reduction catalyst because formationmore » 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 CO{sub 2} reduction at gold nanocluster catalysts.« less

  17. '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

    2014-04-09

    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.

  18. Enhanced enzyme kinetic stability by increasing rigidity within the active site.

    PubMed

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-03-14

    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 Ser(105) 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 T50(15), 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.

  19. GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

    PubMed

    Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

    2017-07-03

    Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Den site activity patterns of adult male and female swift foxes, Vulpes velox, in Northwestern Texas

    USGS Publications Warehouse

    Lemons, P.R.; Ballard, W.B.; Sullivan, R.M.; Sovada, M.A.

    2003-01-01

    Activity of Swift Foxes (Vulpes velox) at den sites was studied in northwestern Texas during pup rearing seasons in 2000 and 2001 to determine role of males in parental care. Twenty-four percent of radio-collared females with a potential to breed successfully raised pups to eight weeks of age. We intensively monitored presence and absence of male and female Swift Foxes at two den sites each year. Females were present >2.6 times more at den sites than males during the pup rearing season. Female and male Swift Foxes largely stayed at dens during diurnal hours and were active away from dens during nocturnal and crepuscular hours. Females and males spent 12.4% and 3.0% more time at dens before pups emerged, than after pups emerged, respectively. Following depredation of one male parent, the female spent 29% less time at the den site. Decrease in time spent at the den by the female following loss of her mate suggested that loss of one parent might severely impact recruitment of Swift Foxes. Our observations indicated that intense Coyote (Canis latrans) depredation may severely impact pup-rearing success as well as the parental care within Swift Fox family groups.

  1. Facebook, Twitter Activities Sites, Location and Students' Interest in Learning

    ERIC Educational Resources Information Center

    Igbo, J. N.; Ezenwaji, Ifeyinwa; Ajuziogu, Christiana U.

    2018-01-01

    This study was carried out to ascertain the influence of social networking sites activities (twitter and Facebook) on secondary school students' interest in learning It also considered the impact of these social networking sites activities on location of the students. Two research questions and two null hypotheses guided the study. Mean and…

  2. Stable isotopes suggest low site fidelity in Bar-Headed Geese (Anser indicus) in Mongolia: Implications for disease transmission

    USGS Publications Warehouse

    Bridge, Eli S.; Kelly, Jeffrey F.; Xiangming Xiao,; Batbayar, Nyambayar; Natsagdorj, Tseveenmyadag; Hill, Nichola J.; Takekawa, John Y.; Hawkes, Lucy A.; Bishop, Charles M.; Butler, Patrick J.; Newman, Scott H.

    2015-01-01

    Population connectivity is an important consideration in studies of disease transmission and biological conservation, especially with regard to migratory species. Determining how and when different subpopulations intermingle during different phases of the annual cycle can help identify important geographical regions or features as targets for conservation efforts and can help inform our understanding of continental-scale disease transmission. In this study, stable isotopes of hydrogen and carbon in contour feathers were used to assess the degree of molt-site fidelity among Bar-headed Geese (Anser indicus) captured in north-central Mongolia. Samples were collected from actively molting Bar-headed Geese (n = 61), and some individual samples included both a newly grown feather (still in sheath) and an old, worn feather from the bird's previous molt (n = 21). Although there was no difference in mean hydrogen isotope ratios for the old and new feathers, the isotopic variance in old feathers was approximately three times higher than that of the new feathers, which suggests that these birds use different and geographically distant molting locations from year to year. To further test this conclusion, online data and modeling tools from the isoMAP website were used to generate probability landscapes for the origin of each feather. Likely molting locations were much more widespread for old feathers than for new feathers, which supports the prospect of low molt-site fidelity. This finding indicates that population connectivity would be greater than expected based on data from a single annual cycle, and that disease spread can be rapid even in areas like Mongolia where Bar-headed Geese generally breed in small isolated groups.

  3. Suggested Activities to Use With Children Who Present Symptoms of Visual Perception Problems, Elementary Level.

    ERIC Educational Resources Information Center

    Washington County Public Schools, Washington, PA.

    Symptoms displayed by primary age children with learning disabilities are listed; perceptual handicaps are explained. Activities are suggested for developing visual perception and perception involving motor activities. Also suggested are activities to develop body concept, visual discrimination and attentiveness, visual memory, and figure ground…

  4. Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

    PubMed

    Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

    2017-07-21

    E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Acquisition of Mathematical Language: Suggestions and Activities for English Language Learners

    ERIC Educational Resources Information Center

    Cirillo, Michelle; Bruna, Katherine Richardson; Herbel-Eisenmann, Beth

    2010-01-01

    In this article, we describe aspects of mathematical language that could be problematic to English-language learners, provide recommendations for teaching English-language learners, and suggest activities intended to foster language development in mathematics. (Contains 1 figure.)

  6. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

    PubMed

    Lee, Ciaran M; Davis, Timothy H; Bao, Gang

    2018-04-01

    What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

  7. Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

    PubMed Central

    Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

    2014-01-01

    Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

  8. Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

    PubMed

    Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

    2014-03-28

    Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

  9. Characterization of the active site properties of CYP4F12.

    PubMed

    Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

    2014-10-01

    Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

    PubMed Central

    Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

    2011-01-01

    In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

  11. Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.

    PubMed Central

    Dominici, P.; Moore, P. S.; Castellani, S.; Bertoldi, M.; Voltattorni, C. B.

    1997-01-01

    Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation. PMID:9300500

  12. Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

    PubMed

    Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

    2016-11-01

    Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

  13. Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.

    PubMed

    Rodrigues, Andria V; Kandegedara, Ashoka; Rotondo, John A; Dancis, Andrew; Stemmler, Timothy L

    2015-06-01

    Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

  14. Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis.

    PubMed

    Bolten, Marcel; Vahlensieck, Christian; Lipp, Colette; Leibundgut, Marc; Ban, Nenad; Weber-Ban, Eilika

    2017-03-10

    Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. [NiFe] hydrogenases: a common active site for hydrogen metabolism under diverse conditions.

    PubMed

    Shafaat, Hannah S; Rüdiger, Olaf; Ogata, Hideaki; Lubitz, Wolfgang

    2013-01-01

    Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these "nonstandard" [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term "oxygen-tolerance". This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency☆

    PubMed Central

    Simon, Mariella T.; Ng, Bobby G.; Friederich, Marisa W.; Wang, Raymond Y.; Boyer, Monica; Kircher, Martin; Collard, Renata; Buckingham, Kati J.; Chang, Richard; Shendure, Jay; Nickerson, Deborah A.; Bamshad, Michael J.; Van Hove, Johan L.K.; Freeze, Hudson H.; Abdenur, Jose E.

    2017-01-01

    We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency. PMID:28216230

  17. Local anesthetics QX 572 and benzocaine act at separate sites on the batrachotoxin-activated sodium channel

    PubMed Central

    1981-01-01

    We have studied the effect of local anesthetics QX 572, which is permanently charged, and benzocaine, which is neutral, on batrachotoxin- activated sodium channels in mouse neuroblastoma N18 cells. The dose- response curves for each drug suggest that QX 752 and benzocaine each act on a single class of binding sites. The dissociation constants are 3.15 X 10(-5) M for QX 572 and 2.65 X 10(-4) M for benzocaine. Equilibrium and kinetic experiments indicate that both drugs are competitive inhibitors of batrachotoxin. When benzocaine and QX 572 are present with batrachotoxin, they are much more effective at inhibiting Na+ flux than would be predicted by a one-site model. Our results indicate that QX 572 and benzocaine bind to separate sites, each of which interacts competitively with batrachotoxin. PMID:6267160

  18. A hybrid QM/MM simulation study of intramolecular proton transfer in the pyridoxal 5'-phosphate in the active site of transaminase: influence of active site interaction on proton transfer.

    PubMed

    Dutta Banik, Sindrila; Chandra, Amalendu

    2014-09-25

    Pyridoxal 5'-phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium that involves an intramolecular proton transfer between the N-protonated zwitterionic ketoenamine tautomer and the O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in the zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP aspartate (PLP-Asp) Schiff base in the active site of aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical simulations. The main focus of the present study is to analyze the factors that control the tautomeric equilibrium in the active sites of various PLP-dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for the ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in the ketoenamine tautomer, while in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP-dependent enzymes (aspartate aminotransferase, Dopa decarboxylase, and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium toward the N

  19. Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

    PubMed

    Fecker, Tobias; Galaz-Davison, Pablo; Engelberger, Felipe; Narui, Yoshie; Sotomayor, Marcos; Parra, Loreto P; Ramírez-Sarmiento, César A

    2018-03-27

    Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic

  20. Generation of 3D templates of active sites of proteins with rigid prosthetic groups.

    PubMed

    Nebel, Jean-Christophe

    2006-05-15

    With the increasing availability of protein structures, the generation of biologically meaningful 3D patterns from the simultaneous alignment of several protein structures is an exciting prospect: active sites could be better understood, protein functions and protein 3D structures could be predicted more accurately. Although patterns can already be generated at the fold and topological levels, no system produces high-resolution 3D patterns including atom and cavity positions. To address this challenge, our research focuses on generating patterns from proteins with rigid prosthetic groups. Since these groups are key elements of protein active sites, the generated 3D patterns are expected to be biologically meaningful. In this paper, we present a new approach which allows the generation of 3D patterns from proteins with rigid prosthetic groups. Using 237 protein chains representing proteins containing porphyrin rings, our method was validated by comparing 3D templates generated from homologues with the 3D structure of the proteins they model. Atom positions were predicted reliably: 93% of them had an accuracy of 1.00 A or less. Moreover, similar results were obtained regarding chemical group and cavity positions. Results also suggested our system could contribute to the validation of 3D protein models. Finally, a 3D template was generated for the active site of human cytochrome P450 CYP17, the 3D structure of which is unknown. Its analysis showed that it is biologically meaningful: our method detected the main patterns of the cytochrome P450 superfamily and the motifs linked to catalytic reactions. The 3D template also suggested the position of a residue, which could be involved in a hydrogen bond with CYP17 substrates and the shape and location of a cavity. Comparisons with independently generated 3D models comforted these hypotheses. Alignment software (Nestor3D) is available at http://www.kingston.ac.uk/~ku33185/Nestor3D.html

  1. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  2. A 5′ Splice Site-Proximal Enhancer Binds SF1 and Activates Exon Bridging of a Microexon

    PubMed Central

    Carlo, Troy; Sierra, Rebecca; Berget, Susan M.

    2000-01-01

    Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3′ and 5′ splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5′ splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3′ splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon. PMID:10805741

  3. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Crichlow, G.; Lubetsky, J; Leng, L

    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 datamore » 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.« less

  4. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity

    PubMed Central

    Cork, David M.W.; Darby, Steven; Ryan-Munden, Claudia A.; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke

    2017-01-01

    Abstract The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway. PMID:27903893

  5. Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity

    PubMed Central

    TRIPATHI, ASHUTOSH; DURRANT, DAVID; LEE, RAY M.; BARUCHELLO, RICCARDO; ROMAGNOLI, ROMEO; SIMONI, DANIELE; KELLOGG, GLEN E.

    2009-01-01

    The crucial role of the microtubule in the cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the αβ-tubulin colchicine site. Computational docking along with HINT score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 μM) such that calculated and measured free energies of binding correlate with an r2 of 0.89 (standard error ± 0.85 kcal mol−1). This correlation suggests that the activity of unknown compounds may be predicted. PMID:19912057

  6. 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.

  7. 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.

  8. Redox-dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide reductase revealed by cyanide binding studies.

    PubMed

    Grönberg, Karin L C; Watmough, Nicholas J; Thomson, Andrew J; Richardson, David J; Field, Sarah J

    2004-04-23

    The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron.

  9. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

    PubMed

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

  10. Mutational analysis of the active site flap (20s loop) of mandelate racemase.

    PubMed

    Bourque, Jennifer R; Bearne, Stephen L

    2008-01-15

    Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG

  11. Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

    PubMed

    Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2018-02-14

    Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

  12. CASPASE-9 CARD:CORE DOMAIN INTERACTIONS REQUIRE A PROPERLY-FORMED ACTIVE SITE

    PubMed Central

    Huber, Kristen L.; Serrano, Banyuhay P.; Hardy, Jeanne A.

    2018-01-01

    Caspase-9 is a critical factor in the initiation of apoptosis, and as a result is tightly regulated by a number of mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Together these findings provide mechanistic insight into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate. PMID:29500231

  13. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

    PubMed Central

    Goto, Asako; Charman, Mark; Ridgway, Neale D.

    2016-01-01

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50–70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

  14. Prediction of active sites of enzymes by maximum relevance minimum redundancy (mRMR) feature selection.

    PubMed

    Gao, Yu-Fei; Li, Bi-Qing; Cai, Yu-Dong; Feng, Kai-Yan; Li, Zhan-Dong; Jiang, Yang

    2013-01-27

    Identification of catalytic residues plays a key role in understanding how enzymes work. Although numerous computational methods have been developed to predict catalytic residues and active sites, the prediction accuracy remains relatively low with high false positives. In this work, we developed a novel predictor based on the Random Forest algorithm (RF) aided by the maximum relevance minimum redundancy (mRMR) method and incremental feature selection (IFS). We incorporated features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure and solvent accessibility to predict active sites of enzymes and achieved an overall accuracy of 0.885687 and MCC of 0.689226 on an independent test dataset. Feature analysis showed that every category of the features except disorder contributed to the identification of active sites. It was also shown via the site-specific feature analysis that the features derived from the active site itself contributed most to the active site determination. Our prediction method may become a useful tool for identifying the active sites and the key features identified by the paper may provide valuable insights into the mechanism of catalysis.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

    The activation of molecular O 2as 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 O 2activation 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 O 2dissociation, 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 O 2dissociationmore » 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 O 2dissociation 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

    2016-01-01

    The activation of molecular O 2as 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 O 2activation 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 O 2dissociation, 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 O 2dissociationmore » 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 O 2dissociation 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V., E-mail: chern@ns.kinetics.nsc.ru

    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 shownmore » 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.« less

  18. Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

    PubMed

    Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

    2017-05-19

    The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

  19. Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

    PubMed

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

  20. 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.

  1. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    PubMed Central

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

  2. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    NASA Astrophysics Data System (ADS)

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

  3. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes

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

    PubMed Central

    Miao, Yinglong; Baudry, Jerome

    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. PMID:21943431

  5. Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

    PubMed Central

    Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh; Wilson, Samuel H.; Mondragón, Alfonso

    2013-01-01

    Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo. PMID:23125368

  6. The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg 2+ ion in the active site and a putative RNA-binding site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Min, Andrew B; Miallau, Linda; Sawaya, Michael R

    VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA.more » The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg2+ ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg2+ ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.« less

  7. Active Site Mutations Change the Cleavage Specificity of Neprilysin

    PubMed Central

    Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

    2012-01-01

    Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

  8. Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

    PubMed

    Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

    1997-02-15

    Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

  9. Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

    PubMed

    Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

    2014-06-01

    Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

  10. An evaluation of the behaviour-change techniques used on Canadian cancer centre Web sites to support physical activity behaviour for breast cancer survivors

    PubMed Central

    Sylvester, B.D.; Zammit, K.; Fong, A.J.; Sabiston, C.M.

    2017-01-01

    Background Cancer centre Web sites can be a useful tool for distributing information about the benefits of physical activity for breast cancer (bca) survivors, and they hold potential for supporting health behaviour change. However, the extent to which cancer centre Web sites use evidence-based behaviour change techniques to foster physical activity behaviour among bca survivors is currently unknown. The aim of our study was to evaluate the presentation of behaviour-change techniques on Canadian cancer centre Web sites to promote physical activity behaviour for bca survivors. Methods All Canadian cancer centre Web sites (n = 39) were evaluated by two raters using the Coventry, Aberdeen, and London–Refined (calo-re) taxonomy of behaviour change techniques and the eEurope 2002 Quality Criteria for Health Related Websites. Descriptive statistics were calculated. Results The most common behaviour change techniques used on Web sites were providing information about consequences in general (80%), suggesting goal-setting behaviour (56%), and planning social support or social change (46%). Overall, Canadian cancer centre Web sites presented an average of M = 6.31 behaviour change techniques (of 40 that were coded) to help bca survivors increase their physical activity behaviour. Evidence of quality factors ranged from 90% (sites that provided evidence of readability) to 0% (sites that provided an editorial policy). Conclusions Our results provide preliminary evidence that, of 40 behaviour-change techniques that were coded, fewer than 20% were used to promote physical activity behaviour to bca survivors on cancer centre Web sites, and that the most effective techniques were inconsistently used. On cancer centre Web sites, health promotion specialists could focus on emphasizing knowledge mobilization efforts using available research into behaviour-change techniques to help bca survivors increase their physical activity. PMID:29270056

  11. An evaluation of the behaviour-change techniques used on Canadian cancer centre Web sites to support physical activity behaviour for breast cancer survivors.

    PubMed

    Sylvester, B D; Zammit, K; Fong, A J; Sabiston, C M

    2017-12-01

    Cancer centre Web sites can be a useful tool for distributing information about the benefits of physical activity for breast cancer (bca) survivors, and they hold potential for supporting health behaviour change. However, the extent to which cancer centre Web sites use evidence-based behaviour change techniques to foster physical activity behaviour among bca survivors is currently unknown. The aim of our study was to evaluate the presentation of behaviour-change techniques on Canadian cancer centre Web sites to promote physical activity behaviour for bca survivors. All Canadian cancer centre Web sites ( n = 39) were evaluated by two raters using the Coventry, Aberdeen, and London-Refined (calo-re) taxonomy of behaviour change techniques and the eEurope 2002 Quality Criteria for Health Related Websites. Descriptive statistics were calculated. The most common behaviour change techniques used on Web sites were providing information about consequences in general (80%), suggesting goal-setting behaviour (56%), and planning social support or social change (46%). Overall, Canadian cancer centre Web sites presented an average of M = 6.31 behaviour change techniques (of 40 that were coded) to help bca survivors increase their physical activity behaviour. Evidence of quality factors ranged from 90% (sites that provided evidence of readability) to 0% (sites that provided an editorial policy). Our results provide preliminary evidence that, of 40 behaviour-change techniques that were coded, fewer than 20% were used to promote physical activity behaviour to bca survivors on cancer centre Web sites, and that the most effective techniques were inconsistently used. On cancer centre Web sites, health promotion specialists could focus on emphasizing knowledge mobilization efforts using available research into behaviour-change techniques to help bca survivors increase their physical activity.

  12. Site preference for luminescent activator ions in doped fluoroperovskite RbZnF3.

    PubMed

    Saroj, Sanjay Kumar; Nagarajan, Rajamani

    2018-08-05

    With the dual objective of investigating the site preferences of larger sized activator ions and to append luminescence property to the perovskite structured RbZnF 3 , doping of manganese(II), cerium(III), europium(III) and terbium(III) ions (5 mol%) was carried out. Although cubic symmetry of RbZnF 3 was preserved for all the doped samples, site preference of rare-earth ions for the A-site Rb + leading to an inverse perovskite arrangement has been noticed from careful analysis of lattice parameters from refinement of powder X-ray diffraction data. Undoped RbZnF 3 exhibited rod-like morphology in the transmission electron microscopic image. In addition to an intense band around 230 nm assignable to the charge transfer from ZnF 3 - to Rb + , typical transitions of respective dopant ions were observed in their UV-visible spectra. The doped samples showed luminescence in blue, green and red regions and time decay experiments suggested uniform dispersion of them without any clustering effect. The lower phonon energy of RbZnF 3 matrix by virtue of the presence of heavier rubidium at the A-site together with its doping with rare-earth ions resulting in an inverse perovskite like arrangement could favour their utility in various practical applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Low erythrocyte Na/K-pump activity and number in northeast Thailand adults: evidence suggesting an acquired disorder.

    PubMed

    Tosukhowong, P; Tungsanga, K; Kittinantavorakoon, C; Chaitachawong, C; Pansin, P; Sriboonlue, P; Sitprija, V

    1996-07-01

    Healthy northeastern Thais have a higher erythrocyte sodium concentration and a lower erythrocyte membrane Na,K-adenosine triphosphatase (ATPase) activity than central Thais. To elucidate whether the defect is hereditary or acquired, we studied plasma sodium and potassium and erythrocyte sodium, potassium, Na,K-ATPase activity, and ouabain-binding sites (OBS) in the following groups: healthy newborns of ethnic central Thais (group 1), healthy newborns of ethnic northeast Thais (group 2), healthy adults of central Thailand ethnicity who lived in the rural central region (group 3) or in Bangkok (group 4), healthy adults of northeast Thailand ethnicity who lived in the rural northeast region (group 5) or who migrated to work in Bangkok for at least 1 year (group 6). Erythrocyte Na was higher in group 2 than in group 1. Group 3 had lower erythrocyte Na,K-ATPase activity than group 4, and it was lower in group 5 than in group 6. Among all groups, group 5 had the highest erythrocyte Na (11.6 mmol/L,F < 0.0001) and the lowest Na,K-ATPase activity (63 mmol Pi/mg x h, F < 0.0001) and erythrocyte OBS (397 sites per cell, F < 0.05) than the other adult groups. There was a positive correlation between erythrocyte Na,K-ATPase and erythrocyte OBS (r = .416, P < .0001). Multiple regression analysis demonstrated a correlation between erythrocyte Na as a dependent variable and erythrocyte OBS, plasma potassium, erythrocyte potassium, and erythrocyte Na,K-ATPase (r = .517, P < .0001). The erythrocyte Na,K-ATPase/OBS ratio, an expression of Na,K-ATPase activity equalized for the number of Na,K-pump units, was lowest among rural adults of the central region (group 3) and the northeast region (group 5) (F < 0.0002). Our data suggest that rural dwellers in Thailand tend to have lower erythrocyte Na,K-ATPase activity than urban dwellers and that this is probably acquired after birth. It was more severe among those from the northeast versus the central region, and was less severe among

  14. Ultrafast Light-Driven Substrate Expulsion from the Active Site of a Photoswitchable Catalyst.

    PubMed

    Pescher, Manuel D; van Wilderen, Luuk J G W; Grützner, Susanne; Slavov, Chavdar; Wachtveitl, Josef; Hecht, Stefan; Bredenbeck, Jens

    2017-09-25

    The photoswitchable piperidine general base catalyst is a prototype structure for light control of catalysis. Its azobenzene moiety moves sterically shielding groups to either protect or expose the active site, thereby changing the basicity and hydrogen-bonding affinity of the compound. The reversible switching dynamics of the catalyst is probed in the infrared spectral range by monitoring hydrogen bond (HB) formation between its active site and methanol (MeOH) as HB donor. Steady-state infrared (IR) and ultrafast IR and UV/Vis spectroscopies are used to uncover ultrafast expulsion of MeOH from the active site within a few picoseconds. Thus, the force generated by the azobenzene moiety even in the final phase of its isomerization is sufficient to break a strong HB within 3 ps and to shut down access to the active site. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

    PubMed

    Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

    2017-02-27

    The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

  16. Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

    PubMed

    Ali, Abid; Azam, Mohd W; Khan, Asad U

    2018-06-01

    New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

    PubMed

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-12-16

    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.

  18. The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site

    PubMed Central

    Fasciglione, Giovanni Francesco; Sbardella, Diego; Sciandra, Francesca; Casella, MariaLuisa; Camerini, Serena; Crescenzi, Marco; Gori, Alessandro; Tarantino, Umberto; Cozza, Paola; Brancaccio, Andrea; Coletta, Massimo; Bozzi, Manuela

    2018-01-01

    Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483–628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621–628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613–651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613–651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor. PMID:29447293

  19. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

    PubMed

    Goto, Asako; Charman, Mark; Ridgway, Neale D

    2016-01-15

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Homology modeling and molecular dynamics simulation of N-myristoyltransferase from protozoan parasites: active site characterization and insights into rational inhibitor design

    NASA Astrophysics Data System (ADS)

    Sheng, Chunquan; Ji, Haitao; Miao, Zhenyuan; Che, Xiaoyin; Yao, Jianzhong; Wang, Wenya; Dong, Guoqiang; Guo, Wei; Lü, Jiaguo; Zhang, Wannian

    2009-06-01

    Myristoyl-CoA:protein N-myristoyltransferase (NMT) is a cytosolic monomeric enzyme that catalyzes the transfer of the myristoyl group from myristoyl-CoA to the N-terminal glycine of a number of eukaryotic cellular and viral proteins. Recent experimental data suggest NMT from parasites could be a promising new target for the design of novel antiparasitic agents with new mode of action. However, the active site topology and inhibitor specificity of these enzymes remain unclear. In this study, three-dimensional models of NMT from Plasmodium falciparum (PfNMT), Leishmania major (LmNMT) and Trypanosoma brucei (TbNMT) were constructed on the basis of the crystal structures of fungal NMTs using homology modeling method. The models were further refined by energy minimization and molecular dynamics simulations. The active sites of PfNMT, LmNMT and TbNMT were characterized by multiple copy simultaneous search (MCSS). MCSS functional maps reveal that PfNMT, LmNMT and TbNMT share a similar active site topology, which is defined by two hydrophobic pockets, a hydrogen-bonding (HB) pocket, a negatively-charged HB pocket and a positively-charged HB pocket. Flexible docking approaches were then employed to dock known inhibitors into the active site of PfNMT. The binding mode, structure-activity relationships and selectivity of inhibitors were investigated in detail. From the results of molecular modeling, the active site architecture and certain key residues responsible for inhibitor binding were identified, which provided insights for the design of novel inhibitors of parasitic NMTs.

  1. Mutations close to a hub residue affect the distant active site of a GH1 β-glucosidase.

    PubMed

    Souza, Valquiria P; Ikegami, Cecília M; Arantes, Guilherme M; Marana, Sandro R

    2018-01-01

    The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the β-glycosidase Sfβgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a β-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfβgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfβgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a β-strand face towards F251, inactivated Sfβgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfβgly active site through short connection pathways along the protein network.

  2. Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*

    PubMed Central

    Mautner, Henry G.; Bartels, Eva

    1970-01-01

    p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholine-receptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or p-nitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptor-activator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue. PMID:5272331

  3. Factors related to physical activity adherence in women: review and suggestions for future research.

    PubMed

    White, Jennifer L; Ransdell, Lynda B; Vener, Jamie; Flohr, Judith A

    2005-01-01

    Approximately 50 percent of individuals who start an exercise program withdraw within 6 months. Thus, many individuals withdraw before health benefits have been realized. This is a disconcerting statistic considering the well known benefits of physical activity for decreasing risk of hypokinetic diseases and improving quality of life. The literature has suggested a plethora of factors to increase the number of individuals who initiate a physical activity program. However, little is known about the factors that keep women exercising-otherwise known as exercise adherence. The purpose of this paper is to: (a) systematically review the quantitative literature to discern the major factors contributing to adherence to physical activity in women and men and make recommendations for specific gender-based considerations that are important when designing PA interventions for women, and (b) suggest areas of future research related to increasing adherence to physical activity in women. Key factors reviewed in this paper may be useful in developing efficacious physical activity programs for women.

  4. Structure-function studies on the active site of the coelenterazine-dependent luciferase from Renilla.

    PubMed

    Woo, Jongchan; Howell, Matthew H; von Arnim, Albrecht G

    2008-04-01

    Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the alpha/beta hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220--three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB--also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate.

  5. Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

    PubMed Central

    Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

    1992-01-01

    By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

  6. Flexibility of active-site gorge aromatic residues and non-gorge aromatic residues in acetylcholinesterase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghattyvenkatakrishna, Pavan K; Uberbacher, Edward C

    2013-01-01

    The presence of an unusually large number of aromatic residues in the active site gorge of acetylcholinesterase has been a topic of great interest. Flexibility of these residues has been suspected to be a key player in controlling ligand traversal in the gorge. This raises the question of whether the over representation of aromatic residues in the gorge implies higher than normal flexibility of those residues. The current study suggests that it does not. Large changes in the hydrophobic cross sectional area due to dihedral oscillations are probably the reason behind their presence in the gorge.

  7. Promoting Physical Activity Participation among Adolescents: The Barriers and the Suggestions

    PubMed Central

    Peykari, Niloofar; Eftekhari, Monir Baradaran; Tehrani, Fahimeh Ramezani; Afzali, Hosein Malek; Hejazi, Farzaneh; Atoofi, Mehrdad Kazemzadeh; Qorbani, Mostafa; Asayesh, Hamid; Djalalinia, Shirin

    2015-01-01

    Background: Physical activity is a complex behavior. To designing the effective intervention, qualitative researches may be allowed for greater understanding of the reasons behind the adolescences’ physical activity-related behaviors’. Methods: Using the grounded theory approach, including semi-structured focus group discussions (FGDs) and in-depth interviews, we conducted a quantitative study to elicit the adolescents and key informants’ opinion regarding the satiation, needs, social and environmental barriers of adolescents’ physical activity. For FGDs, participants were selected from volunteered adolescent (aged 10–19 years) of the populated western part of Tehran, which was selected as a research field. Key informants were invited from the health professionals and experts in the field of adolescents’ health. Results: According to findings, although the majority of participants agreed on the important role of physical activity, the lack of essential motivation and the pressure of educational assignments remove it from the daily program priorities. Lack of a safe environment for girls’ physical activity and high cost of professional sports were two first mentioned barriers. It was also suggested that future interventions should focus on improving more parents’ engagement and their direct participation in physical activities with their adolescents. Conclusions: We proposed the participatory strategies for adolescent's physical activity promotion. Through which target groups participation during the designing, development, and implementation of health programs led to more effective interventions. PMID:25789144

  8. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

    PubMed

    Sayer, Christopher; Finnigan, William; Isupov, Michail N; Levisson, Mark; Kengen, Servé W M; van der Oost, John; Harmer, Nicholas J; Littlechild, Jennifer A

    2016-05-10

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.

  9. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  10. Mapping intermolecular interactions and active site conformations: from human MMP-1 crystal structure to molecular dynamics free energy calculations.

    PubMed

    Nash, Anthony; Birch, Helen L; de Leeuw, Nora H

    2017-02-01

    The zinc-dependent Matrix Metalloproteinases (MMPs) found within the extracellular matrix (ECM) of vertebrates are linked to pathological processes such as arthritis, skin ulceration and cancer. Although a general backbone proteolytic mechanism is understood, crystallographic data continue to suggest an active site that is too narrow to encompass the respective substrate. We present a fully parameterised Molecular Dynamics (MD) study of the structural properties of an MMP-1-collagen crystallographic structure (Protein Data Bank - 4AUO), followed by an exploration of the free energy surface of a collagen polypeptide chain entering the active site, using a combined meta-dynamics and umbrella sampling (MDUS) approach. We conclude that the interactions between MMP-1 and the collagen substrate are in good agreement with a number of experimental studies. As such, our unrestrained MD simulations and our MDUS results, which indicate an energetic barrier for a local uncoiling and insertion event, can inform future investigations of the collagen-peptide non-bonded association steps with the active site prior to proteolytic mechanisms. The elucidation of such free energy barriers provides a better understanding of the role of the enzyme in the ECM and is important in the design of future MMP inhibitors.

  11. Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

    PubMed

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

    2012-04-20

    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.

  12. Synthesis of a molecularly defined single-active site heterogeneous catalyst for selective oxidation of N-heterocycles.

    PubMed

    Zhang, Yujing; Pang, Shaofeng; Wei, Zhihong; Jiao, Haijun; Dai, Xingchao; Wang, Hongli; Shi, Feng

    2018-04-13

    Generally, a homogeneous catalyst exhibits good activity and defined active sites but it is difficult to recycle. Meanwhile, a heterogeneous catalyst can easily be reused but its active site is difficult to reveal. It is interesting to bridge the gap between homogeneous and heterogeneous catalysis via controllable construction of a heterogeneous catalyst containing defined active sites. Here, we report that a molecularly defined, single-active site heterogeneous catalyst has been designed and prepared via the oxidative polymerization of maleimide derivatives. These polymaleimide derivatives can be active catalysts for the selective oxidation of heterocyclic compounds to quinoline and indole via the recycling of -C=O and -C-OH groups, which was confirmed by tracing the reaction with GC-MS using maleimide as the catalyst and by FT-IR analysis with polymaleimide as the catalyst. These results might promote the development of heterogeneous catalysts with molecularly defined single active sites exhibiting a comparable activity to homogeneous catalysts.

  13. Active site proton delivery and the lyase activity of human CYP17A1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbonmore » scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which

  14. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

    PubMed

    Strotmeier, Jasmin; Gu, Shenyan; Jutzi, Stephan; Mahrhold, Stefan; Zhou, Jie; Pich, Andreas; Eichner, Timo; Bigalke, Hans; Rummel, Andreas; Jin, Rongsheng; Binz, Thomas

    2011-07-01

    The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins. © 2011 Blackwell Publishing Ltd.

  15. Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

    PubMed

    Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

    2018-05-09

    Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  16. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase.

    PubMed

    Murphy, Jesse R; Mullins, Elwood A; Kappock, T Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  17. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

    PubMed Central

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  18. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    DOE PAGES

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-05-23

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes andmore » orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.« less

  19. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    PubMed

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  20. Engineering streptokinase for generation of active site-labeled plasminogen analogs*

    PubMed Central

    Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

    2011-01-01

    We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

  1. The biologically active zone in upland habitats at the Hanford Site, Washington, USA: Focus on plant rooting depth and biomobilization.

    PubMed

    Lovtang, Sara; Delistraty, Damon; Rochette, Elizabeth

    2018-07-01

    We challenge the suggestion by Sample et al. (2015) that a depth of 305 cm (10 ft) exceeds the depth of biological activity in soils at the Hanford Site, Washington, USA, or similar sites. Instead, we support the standard point of compliance, identified in the Model Toxics Control Act in the state of Washington, which specifies a depth of 457 cm (15 ft) for the protection of both human and ecological receptors at the Hanford Site. Our position is based on additional information considered in our expanded review of the literature, the influence of a changing environment over time, plant community dynamics at the Hanford Site, and inherent uncertainty in the Sample et al. (2015) analysis. Integr Environ Assess Manag 2018;14:442-446. © 2018 SETAC. © 2018 SETAC.

  2. A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes*

    PubMed Central

    Lopata, Anna; Leveles, Ibolya; Bendes, Ábris Ádám; Viskolcz, Béla; Vértessy, Beáta G.; Jójárt, Balázs; Tóth, Judit

    2016-01-01

    dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent. PMID:27815500

  3. 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

  4. Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

    PubMed Central

    tenBrink, Felix; Schink, Bernhard; Kroneck, Peter M. H.

    2011-01-01

    The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme. PMID:21193613

  5. Comparison of the active-site design of molybdenum oxo-transfer enzymes by quantum mechanical calculations.

    PubMed

    Li, Jilai; Ryde, Ulf

    2014-11-17

    There are three families of mononuclear molybdenum enzymes that catalyze oxygen atom transfer (OAT) reactions, named after a typical example from each family, viz., dimethyl sulfoxide reductase (DMSOR), sulfite oxidase (SO), and xanthine oxidase (XO). These families differ in the construction of their active sites, with two molybdopterin groups in the DMSOR family, two oxy groups in the SO family, and a sulfido group in the XO family. We have employed density functional theory calculations on cluster models of the active sites to understand the selection of molybdenum ligands in the three enzyme families. Our calculations show that the DMSOR active site has a much stronger oxidative power than the other two sites, owing to the extra molybdopterin ligand. However, the active sites do not seem to have been constructed to make the OAT reaction as exergonic as possible, but instead to keep the reaction free energy close to zero (to avoid excessive loss of energy), thereby making the reoxidation (SO and XO) or rereduction of the active sites (DMSOR) after the OAT reaction facile. We also show that active-site models of the three enzyme families can all catalyze the reduction of DMSO and that the DMSOR model does not give the lowest activation barrier. Likewise, all three models can catalyze the oxidation of sulfite, provided that the Coulombic repulsion between the substrate and the enzyme model can be overcome, but for this harder reaction, the SO model gives the lowest activation barrier, although the differences are not large. However, only the XO model can catalyze the oxidation of xanthine, owing to its sulfido ligand.

  6. Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

    PubMed

    Bate, Paul; Warwicker, Jim

    2004-07-02

    Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

  7. 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…

  8. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.

    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, TiO x domains are titrated on TiO x–SiO 2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiO x, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H 2O 2 activation occurs. We use this method to determine the active sitemore » 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.« less

  9. Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

    Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

  10. Identification and characterization of the sodium-binding site of activated protein C.

    PubMed

    He, X; Rezaie, A R

    1999-02-19

    Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

  11. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    PubMed Central

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  12. 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.

  13. 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

  14. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  15. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

    DOE PAGES

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

    2017-07-07

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  16. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tomkinson, B.; Wernstedt, C.; Hellman, U.

    1987-11-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residuesmore » 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.« less

  17. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

    PubMed Central

    Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

  18. 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.

  19. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

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

    2010-01-01

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

  20. Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

    PubMed

    Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B; Bach, Ronald R; Nelsestuen, Gary L

    2005-04-26

    Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

  1. A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

    PubMed

    Kudalkar, Shalley N; Njuma, Olive J; Li, Yongjiang; Muldowney, Michelle; Fuanta, N Rene; Goodwin, Douglas C

    2015-03-03

    Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

  2. 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

  3. Black and Brown Bear Activity at Selected Coastal Sites in Glacier Bay National Park and Preserve, Alaska: A Preliminary Assessment Using Noninvasive Procedures

    USGS Publications Warehouse

    Partridge, Steve; Smith, Tom; Lewis, Tania

    2009-01-01

    A number of efforts in recent years have sought to predict bear activity in various habitats to minimize human disturbance and bear/human conflicts. Alaskan coastal areas provide important foraging areas for bears (Ursus americanus and U. arctos), particularly following den emergence when there may be no snow-free foraging alternatives. Additionally, coastal areas provide important food items for bears throughout the year. Glacier Bay National Park and Preserve (GLBA) in southeastern Alaska has extensive coastal habitats, and the National Park Service (NPS) has been long interested in learning more about the use of these coastal habitats by bears because these same habitats receive extensive human use by park visitors, especially kayaking recreationists. This study provides insight regarding the nature and intensity of bear activity at selected coastal sites within GLBA. We achieved a clearer understanding of bear/habitat relationships within GLBA by analyzing bear activity data collected with remote cameras, bear sign mapping, scat collections, and genetic analysis of bear hair. Although we could not quantify actual levels of bear activity at study sites, agreement among measures of activity (for example, sign counts, DNA analysis, and video record) lends support to our qualitative site assessments. This work suggests that habitat evaluation, bear sign mapping, and periodic scat counts can provide a useful index of bear activity for sites of interest.

  4. Structural analyses of human thymidylate synthase reveal a site that may control conformational switching between active and inactive states.

    PubMed

    Chen, Dan; Jansson, Anna; Sim, Daniel; Larsson, Andreas; Nordlund, Pär

    2017-08-11

    Thymidylate synthase (TS) is the sole enzyme responsible for de novo biosynthesis of thymidylate (TMP) and is essential for cell proliferation and survival. Inhibition of human TS (hTS) has been extensively investigated for cancer chemotherapy, but several aspects of its activity and regulation are still uncertain. In this study, we performed comprehensive structural and biophysical studies of hTS using crystallography and thermal shift assay and provided the first detailed structural information on the conformational changes induced by ligand binding to the hTS active site. We found that upon binding of the antifolate agents raltitrexed and nolatrexed, the two insert regions in hTS, the functions of which are unclear, undergo positional shifts toward the catalytic center. We investigated the inactive conformation of hTS and found that the two insert regions are also involved in the conformational transition between the active and inactive state of hTS. Moreover, we identified a ligand-binding site in the dimer interface, suggesting that the cavity in the dimer interface could serve as an allosteric site of hTS to regulate the conformational switching between the active and inactive states. On the basis of these findings, we propose a regulatory mechanism of hTS activity that involves allosteric regulation of interactions of hTS with its own mRNA depending on cellular demands for TMP. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  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. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. 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.

  7. 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

  8. Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

    PubMed

    Sen, Kakali; Horrell, Sam; Kekilli, Demet; Yong, Chin W; Keal, Thomas W; Atakisi, Hakan; Moreau, David W; Thorne, Robert E; Hough, Michael A; Strange, Richard W

    2017-07-01

    Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

  9. Preseismic Velocity Changes Observed from Active Source Monitoringat the Parkfield SAFOD Drill Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Daley, Thomas; Niu, Fenglin; Silver, Paul G.

    2008-06-10

    Measuring stress changes within seismically active fault zones has been a long-sought goal of seismology. Here we show that such stress changes are measurable by exploiting the stress dependence of seismic wave speed from an active source cross-well experiment conducted at the SAFOD drill site. Over a two-month period we observed an excellent anti-correlation between changes in the time required for an S wave to travel through the rock along a fixed pathway--a few microseconds--and variations in barometric pressure. We also observed two large excursions in the traveltime data that are coincident with two earthquakes that are among those predictedmore » to produce the largest coseismic stress changes at SAFOD. Interestingly, the two excursions started approximately 10 and 2 hours before the events, respectively, suggesting that they may be related to pre-rupture stress induced changes in crack properties, as observed in early laboratory studies.« less

  10. Pharmacy Students' Perceptions of Tobacco Sales in Pharmacies and Suggested Strategies for Promoting Tobacco-Free Experiential Sites

    PubMed Central

    Hudmon, Karen Suchanek; Hussar, Daniel A.; Fenlon, Christine M.; Corelli, Robin L.

    2006-01-01

    Objective The objectives of this report were to estimate the extent to which pharmacy student perceptions are aligned with the 2003 resolution of the American Association of Colleges of Pharmacy (AACP) addressing the use of experiential sites that sell cigarettes and other tobacco products. Methods Pharmacy students participating in a national tobacco cessation training program completed posttraining survey instruments and indicated their opinion about tobacco sales in pharmacies. Responses were examined with respect to students' sex and tobacco use status. Results Of 3,064 students, 3.5% were in favor of tobacco sales in pharmacies. Opinions varied by students' sex (p < 0.001) and tobacco use status (p < 0.001); in logistic regression analyses, males (OR = 2.62; 95% CI, 1.77, 3.89) and current tobacco users (OR = 2.31; 95% CI, 1.41, 3.76) were most likely to be in favor of tobacco sales. Conclusion Few pharmacy students are in favor of tobacco sales in pharmacies. Given the overall lack of support, and acting in accordance with the 2003 AACP resolution, pharmacy schools are encouraged to use only experiential sites that do not sell tobacco products. Suggested strategies for moving toward this goal are presented. PMID:17136194

  11. A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

    PubMed

    Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

    2018-06-01

    The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

    PubMed

    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.

  13. [Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

    PubMed

    Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

    2017-08-25

    Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

  14. Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

    PubMed

    Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

    2013-08-21

    The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

  15. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    PubMed

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

  16. Well-Defined Metal-O6 in Metal-Catecholates as a Novel Active Site for Oxygen Electroreduction.

    PubMed

    Liu, Xuan-He; Hu, Wei-Li; Jiang, Wen-Jie; Yang, Ya-Wen; Niu, Shuai; Sun, Bing; Wu, Jing; Hu, Jin-Song

    2017-08-30

    Metal-nitrogen coordination sites, M-N x (M = Fe, Co, Ni, etc.), have shown great potential to replace platinum group materials as electrocatalysts for oxygen reduction reaction (ORR). However, the real active site in M-N x is still vague to date due to their complicated structure and composition. It is therefore highly desirable but challenging to develop ORR catalysts with novel and clear active sites, which could meet the needs of comprehensive understanding of structure-function relationships and explore new cost-effective and efficient ORR electrocatalysts. Herein, well-defined M-O 6 coordination in metal-catecholates (M-CATs, M = Ni or Co) is discovered to be catalytically active for ORR via a four-electron-dominated pathway. In view of no pyrolysis involved and unambiguous crystalline structure of M-CATs, the M-O 6 octahedral coordination site with distinct structure is determined as a new type of active site for ORR. These findings extend the scope of metal-nonmetal coordination as an active site for ORR and pave a way for bottom-up design of novel electrocatalysts containing M-O 6 coordination.

  17. The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

    PubMed

    Skov, Lars K; Pizzut-Serin, Sandra; Remaud-Simeon, Magali; Ernst, Heidi A; Gajhede, Michael; Mirza, Osman

    2013-09-01

    Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

  18. 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.

  19. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

  20. Probing the Active Surface Sites for CO Reduction on Oxide-Derived Copper Electrocatalysts

    DOE PAGES

    Verdaguer-Casadevall, Arnau; Li, Christina W.; Johansson, Tobias P.; ...

    2015-07-30

    CO electroreduction activity on oxide-derived Cu (OD-Cu) was found to correlate with metastable surface features that bind CO strongly. OD-Cu electrodes prepared by H 2 reduction of Cu 2O precursors reduce CO to acetate and ethanol with nearly 50% Faradaic efficiency at moderate overpotential. Temperature-programmed desorption of CO on OD-Cu revealed the presence of surface sites with strong CO binding that are distinct from the terraces and stepped sites found on polycrystalline Cu foil. After annealing at 350 °C, the surface-area corrected current density for CO reduction is 44-fold lower and the Faradaic efficiency is less than 5%. These changesmore » are accompanied by a reduction in the proportion of strong CO binding sites. Here, we propose that the active sites for CO reduction on OD-Cu surfaces are strong CO binding sites that are supported by grain boundaries. Uncovering these sites is a first step toward understanding the surface chemistry necessary for efficient CO electroreduction.« less

  1. Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

    PubMed Central

    Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W. H.; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

    2010-01-01

    Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, 163DVD165 and 221FWGWGREDDE230, are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp224 as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp228 acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics. PMID:20843813

  2. A DFT Study of Tungsten-Methylidene Formation on a W/ZSM-5 Zeolite: The Metathesis Active Site.

    PubMed

    Maihom, Thana; Probst, Michael; Limtrakul, Jumras

    2015-10-26

    Tungsten-methylidene formation from ethene on either the W(IV) , W(V) , or W(VI) active sites of a W/ZSM-5 zeolite is investigated by using the M06-L functional. The reaction is assumed to proceed in two steps; the first step is the [2+2] cycloaddition between ethene and the W-O active site to form an oxametallacycle intermediate. The intermediate is then decomposed to produce the W-methylidene active site from the metathesis reaction. The overall activation barrier of the reaction on W(VI) (27.3 kcal mol(-1) ) is considerably lower than the ones for W(IV) and W(V) (69.4 and 37.1 kcal mol(-1) , respectively). Moreover, the reaction involving the W(VI) site also stabilizes intermediates and products to a larger extent than the ones on the W(IV) and W(V) sites. As a result, we have demonstrated that the reaction of the W-methylidene metathesis active site is both kinetically and thermodynamically favored to occur on the W(VI) active site of the zeolite. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.

    Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

  4. Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

    DOE PAGES

    Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.; ...

    2018-02-05

    Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

  5. pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

    PubMed

    Hjörleifsson, Jens G; Ásgeirsson, Bjarni

    2017-09-26

    The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7-10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the K i for inorganic phosphate (product inhibition) and the K M for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.

  6. Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

    PubMed

    Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

    2018-05-30

    Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

  7. Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

    PubMed Central

    Lill, R; Robertson, J M; Wintermeyer, W

    1989-01-01

    A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA. PMID:2583120

  8. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

  9. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

    DOE PAGES

    O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

    2016-12-22

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

  10. Molecular investigation of active binding site of isoniazid (INH) and insight into resistance mechanism of S315T-MtKatG in Mycobacterium tuberculosis.

    PubMed

    Srivastava, Gaurava; Tripathi, Shubhandra; Kumar, Akhil; Sharma, Ashok

    2017-07-01

    Multi drug resistant tuberculosis is a major threat for mankind. Resistance against Isoniazid (INH), targeting MtKatG protein, is one of the most commonly occurring resistances in MDR TB strains. S315T-MtKatG mutation is widely reported for INH resistance. Despite having knowledge about the mechanism of INH, exact binding site of INH to MtKatG is still uncertain and proposed to have three presumable binding sites (site-1, site-2, and site-3). In the current study docking, molecular dynamics simulation, binding free energy estimation, principal component analysis and free energy landscape analysis were performed to get molecular level details of INH binding site on MtKatG, and to probe the effect of S315T mutation on INH binding. Molecular docking and MD analysis suggested site-1 as active binding site of INH, where the effects of S315T mutation were observed on both access tunnel as well as molecular interaction between INH and its neighboring residues. MMPBSA also supported site-1 as potential binding site with lowest binding energy of -44.201 kJ/mol. Moreover, PCA and FEL revealed that S315T mutation not only reduces the dimension of heme access tunnel but also showed that extra methyl group at 315 position altered heme cavity, enforcing heme group distantly from INH, and thus preventing INH activation. The present study not only investigated the active binding site of INH but also provides a new insight about the conformational changes in the binding site of S315T-MtKatG. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Molecular dynamics simulation of the last step of a catalytic cycle: product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis.

    PubMed

    Choutko, Alexandra; van Gunsteren, Wilfred F

    2012-11-01

    The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site. Copyright © 2012 The Protein Society.

  12. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... DEPARTMENT OF ENERGY Reimbursement for Costs of Remedial Action at Active Uranium and Thorium...) acceptance of claims in FY 2011 from eligible active uranium and thorium processing site licensees for... incurred by licensees at active uranium and thorium processing sites to remediate byproduct material...

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

    DOE PAGES

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; ...

    2015-03-25

    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. Here, we mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation ofmore » 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. In conclusion, the overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.

    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. Here, we mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation ofmore » 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. In conclusion, the overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.« less

  15. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

    PubMed

    Meyer, Thomas; Melin, Frédéric; Richter, Oliver-M H; Ludwig, Bernd; Kannt, Aimo; Müller, Hanne; Michel, Hartmut; Hellwig, Petra

    2015-02-27

    Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

    PubMed

    Krajewska, Barbara; Zaborska, Wiesława

    2007-10-01

    In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

  17. Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

    PubMed Central

    Gorres, Kelly L.; Pua, Khian Hong; Raines, Ronald T.

    2009-01-01

    The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change. PMID:19890397

  18. 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.

  19. Identification of Tyrosine 972 as a Novel Site of Jak2 Tyrosine Kinase Phosphorylation and its Role in Jak2 Activation

    PubMed Central

    McDoom, Issam; Ma, Xianyue; Kirabo, Annet; Lee, Kuang-Yung; Ostrov, David A.; Sayeski, Peter P.

    2013-01-01

    Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation, and hence auto-regulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild type Jak2 or a full length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2-Y972F mutant exhibited a moderately impaired level of activation when compared to wild type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to activate was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation. PMID:18636744

  20. Transfer hydrogenation over sodium-modified ceria: Enrichment of redox sites active for alcohol dehydrogenation

    DOE PAGES

    Nelson, Nicholas C.; Boote, Brett W.; Naik, Pranjali; ...

    2017-01-17

    Ceria (CeO 2) and sodium-modified ceria (Ce-Na) were prepared through combustion synthesis. Palladium was deposited onto the supports (Pd/CeO 2 and Pd/Ce-Na) and their activity for the aqueous-phase transfer hydrogenation of phenol using 2-propanol under liquid flow conditions was studied. Pd/Ce-Na showed a marked increase (6×) in transfer hydrogenation activity over Pd/CeO 2. Material characterization indicated that water-stable sodium species were not doped into the ceria lattice, but rather existed as subsurface carbonates. Modification of ceria by sodium provided more adsorption and redox active sites (i.e. defects) for 2-propanol dehydrogenation. This effect was an intrinsic property of the Ce-Na supportmore » and independent of Pd. The redox sites active for 2-propanol dehydrogenation were thermodynamically equivalent on both supports/catalysts. At high phenol concentrations, the reaction was limited by 2-propanol adsorption. Furthermore, the difference in catalytic activity was attributed to the different numbers of 2-propanol adsorption and redox active sites on each catalyst.« less

  1. 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. 2009 Elsevier Inc. All rights reserved.

  2. Improved ethanol electrooxidation performance by shortening Pd-Ni active site distance in Pd-Ni-P nanocatalysts

    NASA Astrophysics Data System (ADS)

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-01

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (~5 nm) Pd-Ni-P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni-P heterodimers into Pd-Ni-P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.

  3. Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

    PubMed Central

    Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

    2011-01-01

    Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

  4. A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

    PubMed

    Jacewicz, Agata; Trzemecka, Anna; Guja, Kip E; Plochocka, Danuta; Yakubovskaya, Elena; Bebenek, Anna; Garcia-Diaz, Miguel

    2013-01-01

    Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

  5. Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota.

    PubMed

    Makarova, Alena V; Ignatov, Artem; Miropolskaya, Nataliya; Kulbachinskiy, Andrey

    2014-10-01

    Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn(2+) ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson-Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg(2+) reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson-Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

    PubMed

    Ibrahim, Firas; Andre, Claire; Iutzeler, Anne; Guillaume, Yves Claude

    2013-10-01

    A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k(cat)) was increased while the Michaelis constant (K(m)) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.

  7. Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis.

    PubMed

    Bansal, Vibha; Delgado, Yamixa; Legault, Marc; Barletta, Gabriel

    2012-02-14

    The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

  8. Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis.

    PubMed

    Mathupala, S P; Lowe, S E; Podkovyrov, S M; Zeikus, J G

    1993-08-05

    The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

  9. Sequence features of viral and human Internal Ribosome Entry Sites predictive of their activity

    PubMed Central

    Elias-Kirma, Shani; Nir, Ronit; Segal, Eran

    2017-01-01

    Translation of mRNAs through Internal Ribosome Entry Sites (IRESs) has emerged as a prominent mechanism of cellular and viral initiation. It supports cap-independent translation of select cellular genes under normal conditions, and in conditions when cap-dependent translation is inhibited. IRES structure and sequence are believed to be involved in this process. However due to the small number of IRESs known, there have been no systematic investigations of the determinants of IRES activity. With the recent discovery of thousands of novel IRESs in human and viruses, the next challenge is to decipher the sequence determinants of IRES activity. We present the first in-depth computational analysis of a large body of IRESs, exploring RNA sequence features predictive of IRES activity. We identified predictive k-mer features resembling IRES trans-acting factor (ITAF) binding motifs across human and viral IRESs, and found that their effect on expression depends on their sequence, number and position. Our results also suggest that the architecture of retroviral IRESs differs from that of other viruses, presumably due to their exposure to the nuclear environment. Finally, we measured IRES activity of synthetically designed sequences to confirm our prediction of increasing activity as a function of the number of short IRES elements. PMID:28922394

  10. Work-site wellness programmes in Sweden: a cross-sectional study of physical activity, self-efficacy, and health.

    PubMed

    Gånedahl, H; Zsaludek Viklund, P; Carlén, K; Kylberg, E; Ekberg, J

    2015-05-01

    In Sweden, a work-site wellness programme implies reimbursing some of the expenses for health-promoting activities. Although work-site wellness programmes are readily available in Sweden, a large number of employees elect not to participate. The aim of this study was to investigate the association of physical activity, self-reported general health assessment and self-efficacy with participation in a work-site wellness programme. A cross-sectional study design was used. An online questionnaire was distributed to employees of a manufacturing company with 2500 employees in southwest Sweden. Those who took advantage of the work-site wellness programme assessed their general health as better and had higher assessment of physical activity. The study showed that being enlisted also implies a higher level of physical activity and general health; however, the effect sizes of these correlations were small. Self-efficacy, i.e. perceived behavioural control, was not associated with participation in the work-site wellness programme. However, self-efficacy was correlated with both general health assessment and physical activity. A regression analysis to determine explanatory contributions to the general health assessment score showed no significant contribution from participation in a work-site wellness programme, but was instead explained by perceived behavioural control and physical activity. Given the small effect size of the difference in physical activity between participators and non-participators in the work-site wellness programme, it is probable that only a small proportion of participators changed their health-promoting activities as a result of the work-site wellness programme. Copyright © 2015 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

  11. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    DOE PAGES

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH boundmore » structures have the highest calculated activity to date.« less

  12. The Diverse AAA+ Machines that Repair Inhibited Rubisco Active Sites

    PubMed Central

    Mueller-Cajar, Oliver

    2017-01-01

    Gaseous carbon dioxide enters the biosphere almost exclusively via the active site of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This highly conserved catalyst has an almost universal propensity to non-productively interact with its substrate ribulose 1,5-bisphosphate, leading to the formation of dead-end inhibited complexes. In diverse autotrophic organisms this tendency has been counteracted by the recruitment of dedicated AAA+ (ATPases associated with various cellular activities) proteins that all use the energy of ATP hydrolysis to remodel inhibited Rubisco active sites leading to release of the inhibitor. Three evolutionarily distinct classes of these Rubisco activases (Rcas) have been discovered so far. Green and red-type Rca are mostly found in photosynthetic eukaryotes of the green and red plastid lineage respectively, whereas CbbQO is associated with chemoautotrophic bacteria. Ongoing mechanistic studies are elucidating how the various motors are utilizing both similar and contrasting strategies to ultimately perform their common function of cracking the inhibited Rubisco active site. The best studied mechanism utilized by red-type Rca appears to involve transient threading of the Rubisco large subunit C-terminal peptide, reminiscent of the action performed by Clp proteases. As well as providing a fascinating example of convergent molecular evolution, Rca proteins can be considered promising crop-improvement targets. Approaches aiming to replace Rubisco in plants with improved enzymes will need to ensure the presence of a compatible Rca protein. The thermolability of the Rca protein found in crop plants provides an opportunity to fortify photosynthesis against high temperature stress. Photosynthesis also appears to be limited by Rca when light conditions are fluctuating. Synthetic biology strategies aiming to enhance the autotrophic CO2 fixation machinery will need to take into consideration the requirement for Rubisco activases

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

    PubMed Central

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

    2014-01-01

    Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η1-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η1) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds. PMID:24344312

  14. MX Siting Investigation. Water Resources Program Industry Activity Inventory, Nevada-Utah.

    DTIC Science & Technology

    1980-09-02

    sites. New and revived mining activities and the cooling needs of possible new coal -fired electric power plants represent the chief competitors with MX...34 !- ---- ON CO. Figure .-. Ma showing araipce yUaIoto f𔃻XMsieCmlx 1 3 include new mining activity and coal -fired, geothermal, and hydroelectric j energy...in northeastern Juab County. The Soil Conservation Service has been actively pushing land treatment programs to increase the productivity of irrigated

  15. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mgmore » protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.« less

  16. Enthalpic Breakdown of Water Structure on Protein Active-Site Surfaces

    PubMed Central

    Haider, Kamran; Wickstrom, Lauren; Ramsey, Steven; Gilson, Michael K.; Kurtzman, Tom

    2016-01-01

    The principles underlying water reorganization around simple non-polar solutes are well understood and provide the framework for classical hydrophobic effect, whereby water molecules structure themselves around solutes so that they maintain favorable energetic contacts with both the solute and with other water molecules. However, for certain solute surface topographies, water molecules, due to their geometry and size, are unable to simultaneously maintain favorable energetic contacts with both the surface and neighboring water molecules. In this study, we analyze the solvation of ligand-binding sites for six structurally diverse proteins using hydration site analysis and measures of local water structure, in order to identify surfaces at which water molecules are unable to structure themselves in a way that maintains favorable enthalpy relative to bulk water. These surfaces are characterized by a high degree of enclosure, weak solute-water interactions, and surface constraints that induce unfavorable pair interactions between neighboring water molecules. Additionally, we find that the solvation of charged side-chains in an active site generally results in favorable enthalpy but can also lead to pair interactions between neighboring water molecules that are significantly unfavorable relative to bulk water. We find that frustrated local structure can occur not only in apolar and weakly polar pockets, where overall enthalpy tends to be unfavorable, but also in charged pockets, where overall water enthalpy tends to be favorable. The characterization of local water structure in these terms may prove useful for evaluating the displacement of water from diverse protein active-site environments. PMID:27169482

  17. Nevada National Security Site Industrial Sites Project Closeout - 12498

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cabble, Kevin; Krauss, Mark; Matthews, Pat

    The U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office is responsible for environmental restoration (ER) at the Nevada National Security Site (NNSS). This includes remediation at Industrial Sites where past nuclear testing activities and activities that supported nuclear testing may have or are known to have resulted in the release of contaminants into the environment. Industrial Sites at the NNSS have included nuclear facilities that supported the nuclear rocket/missile development programs, gas stations, landfills, spill sites, ordnance sites, and numerous other waste disposal and release sites. The NNSS Industrial Sites activities neared completion at the endmore » of fiscal year 2011 while other activities required under the Federal Facility Agreement and Consent Order (FFACO) and part of the same NNSS ER Project are forecasted to extend to 2027 or beyond. With the majority of Industrial Sites corrective action units (CAUs) completed (more than 250 CAUs and over 1,800 corrective action sites), it was determined that an activity closeout process should be implemented to ensure that the work completed over the past 15 years is well documented in a comprehensive and concise summary. While the process used to close each individual CAU is described in approved documents, no single document describes in summary fashion the work completed to close the many individual Industrial Sites. The activity closeout process will be used to develop an Industrial Sites closeout document that describes these years of work. This document will summarize the number of Industrial Sites closed under the FFACO and provide general descriptions of projects, contaminants removed, and sites closed in place with corresponding Use Restrictions. Other pertinent information related to Industrial Sites work such as the project history, closure decisions, historical declarations, remediation strategies, and final CAU status will be included in the

  18. Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ.

    PubMed

    Chertkov, O V; Armeev, G A; Uporov, I V; Legotsky, S A; Sykilinda, N N; Shaytan, A K; Klyachko, N L; Miroshnikov, K A

    2017-01-01

    Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C -terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

  19. Active sites for CO 2 hydrogenation to methanol on Cu/ZnO catalysts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kattel, Shyam; Ramírez, Pedro J.; Chen, Jingguang G.

    The active sites over commercial copper/zinc oxide/aluminum oxide (Cu/ZnO/Al 2O 3) catalysts for carbon dioxide (CO 2) hydrogenation to methanol, the Zn-Cu bimetallic sites or ZnO-Cu interfacial sites, have recently been the subject of intense debate. Here, we report a direct comparison between the activity of ZnCu and ZnO/Cu model catalysts for methanol synthesis. By combining x-ray photoemission spectroscopy, density functional theory, and kinetic Monte Carlo simulations, we can identify and characterize the reactivity of each catalyst. Both experimental and theoretical results agree that ZnCu undergoes surface oxidation under the reaction conditions so that surface Zn transforms into ZnO andmore » allows ZnCu to reach the activity of ZnO/Cu with the same Zn coverage. These results highlight a synergy of Cu and ZnO at the interface that facilitates methanol synthesis via formate intermediates.« less

  20. Active sites for CO 2 hydrogenation to methanol on Cu/ZnO catalysts

    DOE PAGES

    Kattel, Shyam; Ramírez, Pedro J.; Chen, Jingguang G.; ...

    2017-03-23

    The active sites over commercial copper/zinc oxide/aluminum oxide (Cu/ZnO/Al 2O 3) catalysts for carbon dioxide (CO 2) hydrogenation to methanol, the Zn-Cu bimetallic sites or ZnO-Cu interfacial sites, have recently been the subject of intense debate. Here, we report a direct comparison between the activity of ZnCu and ZnO/Cu model catalysts for methanol synthesis. By combining x-ray photoemission spectroscopy, density functional theory, and kinetic Monte Carlo simulations, we can identify and characterize the reactivity of each catalyst. Both experimental and theoretical results agree that ZnCu undergoes surface oxidation under the reaction conditions so that surface Zn transforms into ZnO andmore » allows ZnCu to reach the activity of ZnO/Cu with the same Zn coverage. These results highlight a synergy of Cu and ZnO at the interface that facilitates methanol synthesis via formate intermediates.« less

  1. Influence of a Confined Methanol Solvent on the Reactivity of Active Sites in UiO-66.

    PubMed

    Caratelli, Chiara; Hajek, Julianna; Rogge, Sven M J; Vandenbrande, Steven; Meijer, Evert Jan; Waroquier, Michel; Van Speybroeck, Veronique

    2018-02-19

    UiO-66, composed of Zr-oxide bricks and terephthalate linkers, is currently one of the most studied metal-organic frameworks due to its exceptional stability. Defects can be introduced in the structure, creating undercoordinated Zr atoms which are Lewis acid sites. Here, additional Brønsted sites can be generated by coordinated protic species from the solvent. In this Article, a multilevel modeling approach was applied to unravel the effect of a confined methanol solvent on the active sites in UiO-66. First, active sites were explored with static periodic density functional theory calculations to investigate adsorption of water and methanol. Solvent was then introduced in the pores with grand canonical Monte Carlo simulations, followed by a series of molecular dynamics simulations at operating conditions. A hydrogen-bonded network of methanol molecules is formed, allowing the protons to shuttle between solvent methanol, adsorbed water, and the inorganic brick. Upon deprotonation of an active site, the methanol solvent aids the transfer of protons and stabilizes charged configurations via hydrogen bonding, which could be crucial in stabilizing reactive intermediates. The multilevel modeling approach adopted here sheds light on the important role of a confined solvent on the active sites in the UiO-66 material, introducing dynamic acidity in the system at finite temperatures by which protons may be easily shuttled from various positions at the active sites. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  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. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    PubMed

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC 50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - C ata L ytic A ctive S ite P rediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro

  5. The role of the peripheral benzodiazepine receptor in photodynamic activity of certain pyropheophorbide ether photosensitizers: albumin site II as a surrogate marker for activity.

    PubMed

    Dougherty, Thomas J; Sumlin, Adam B; Greco, William R; Weishaupt, Kenneth R; Vaughan, Lurine A; Pandey, Ravindra K

    2002-07-01

    A study has been carried out to define the importance of the peripheral benzodiazepine receptor (PBR) as a binding site for a series of chlorin-type photosensitizers, pyropheophorbide-a ethers, the subject of a previous quantitative structure-activity relationship study by us. The effects of the PBR ligand PK11195 on the photodynamic activity have been determined in vivo for certain members of this series of alkyl-substituted ethers: two of the most active derivatives (hexyl and heptyl), the least active derivative (dodecyl [C12]) and one of intermediate activity (octyl [C8]). The photodynamic therapy (PDT) effect was inhibited by PK11195 for both of the most active derivatives, but no effect on PDT activity was found for the less active C12 or C8 ethers. The inhibitory effects of PK11195 were predicted by the binding of only the active derivatives to the benzodiazepine site on albumin, ie. human serum albumin (HSA)-Site II. Thus, as with certain other types of photosensitizers, it has been demonstrated with this series of pyropheophorbide ethers that in vitro binding to HSA-Site II is a predictor of both optimal in vivo activity and binding to the PBR in vivo.

  6. Molecular Dynamics Study of Twister Ribozyme: Role of Mg(2+) Ions and the Hydrogen-Bonding Network in the Active Site.

    PubMed

    Ucisik, Melek N; Bevilacqua, Philip C; Hammes-Schiffer, Sharon

    2016-07-12

    The recently discovered twister ribozyme is thought to utilize general acid-base catalysis in its self-cleavage mechanism, but the roles of nucleobases and metal ions in the mechanism are unclear. Herein, molecular dynamics simulations of the env22 twister ribozyme are performed to elucidate the structural and equilibrium dynamical properties, as well as to examine the role of Mg(2+) ions and possible candidates for the general base and acid in the self-cleavage mechanism. The active site region and the ends of the pseudoknots were found to be less mobile than other regions of the ribozyme, most likely providing structural stability and possibly facilitating catalysis. A purported catalytic Mg(2+) ion and the closest neighboring Mg(2+) ion remained chelated and relatively immobile throughout the microsecond trajectories, although removal of these Mg(2+) ions did not lead to any significant changes in the structure or equilibrium motions of the ribozyme on the microsecond time scale. In addition, a third metal ion, a Na(+) ion remained close to A1(O5'), the leaving group atom, during the majority of the microsecond trajectories, suggesting that it might stabilize the negative charge on A1(O5') during self-cleavage. The locations of these cations and their interactions with key nucleotides in the active site suggest that they may be catalytically relevant. The P1 stem is partially melted at its top and bottom in the crystal structure and further unwinds in the trajectories. The simulations also revealed an interconnected network comprised of hydrogen-bonding and π-stacking interactions that create a relatively rigid network around the self-cleavage site. The nucleotides involved in this network are among the highly conserved nucleotides in twister ribozymes, suggesting that this interaction network may be important to structure and function.

  7. Male Adolescents' Reasons for Participating in Physical Activity, Barriers to Participation, and Suggestions for Increasing Participation

    ERIC Educational Resources Information Center

    Allison, Kenneth R.; Dwyer, John J. M.; Goldenberg, Ellie; Fein, Allan; Yoshida, Karen K.; Boutilier, Marie

    2005-01-01

    This study explored male adolescents' reasons for participating in moderate and vigorous physical activity, perceived barriers to moderate and vigorous physical activity, and suggestions as to what can be done to increase participation in physical activity. A total of 26 male 15- and 16-year-old adolescents participated in focus group sessions,…

  8. Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

    PubMed Central

    Deng, Hua

    2017-01-01

    Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

  9. Improved ethanol electrooxidation performance by shortening Pd–Ni active site distance in Pd–Ni–P nanocatalysts

    PubMed Central

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-01

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (∼5 nm) Pd–Ni–P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni–P heterodimers into Pd–Ni–P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites. PMID:28071650

  10. Dithiolato-bridged nickel-iron complexes as models for the active site of [NiFe]-hydrogenases.

    PubMed

    Song, Li-Cheng; Yang, Xi-Yue; Cao, Meng; Gao, Xiu-Yun; Liu, Bei-Bei; Zhu, Liang; Jiang, Feng

    2017-03-30

    The structural and functional modeling of the active site of [NiFe]-hydrogenases has been proved to be challenging to a great extent. Herein, we report the synthesis, structures, and some properties of the NiFe-based dicarbonyl, terminal hydride, and μ-hydroxo models for the active site of [NiFe]-hydrogenases.

  11. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

    PubMed

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  12. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model

    PubMed Central

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  13. 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…

  14. Surface Sites for Engineering Allosteric Control in Proteins

    PubMed Central

    Lee, Jeeyeon; Natarajan, Madhusudan; Nashine, Vishal C.; Socolich, Michael; Vo, Tina; Russ, William P.; Benkovic, Stephen J.; Ranganathan, Rama

    2010-01-01

    Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites. PMID:18927392

  15. Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

    PubMed Central

    Ganter, U M; Longstaff, C; Pajares, M A; Rando, R R; Siebert, F

    1991-01-01

    Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species. PMID:2049524

  16. The Way of the Web: Answers to Your Questions about Web Site Marketing.

    ERIC Educational Resources Information Center

    Wassom, Julie

    2002-01-01

    Provides suggestions for effective web site marketing for child care and early education programs. Includes key considerations in designing a web site, specific elements that cause visitors to stay on and return to the site, use of interactive sites, web-site updating and revision, and use of traditional marketing activities to direct prospective…

  17. Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

    PubMed Central

    Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

    2008-01-01

    SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

  18. How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

    PubMed Central

    Hytönen, Vesa P; Vogel, Viola

    2008-01-01

    Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

  19. X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

    PubMed

    Law, Ruby H P; Wu, Guojie; Leung, Eleanor W W; Hidaka, Koushi; Quek, Adam J; Caradoc-Davies, Tom T; Jeevarajah, Devadharshini; Conroy, Paul J; Kirby, Nigel M; Norton, Raymond S; Tsuda, Yuko; Whisstock, James C

    2017-05-09

    The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO ( trans -4-aminomethylcyclohexanecarbonyl-l-tyrosine- n -octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.

  20. Identification of the fatty acid activation site on human ClC-2.

    PubMed

    Cuppoletti, John; Tewari, Kirti P; Chakrabarti, Jayati; Malinowska, Danuta H

    2017-06-01

    Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl - channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET 691 , of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP] i ), EP 2 , or EP 4 receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl - current activation (increasing EC 50 s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA 655 /RGEA 691 ) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP] i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl - currents without increasing [cAMP] i, while PGE 2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP 2 or EP 4 receptors. L-161,982, a supposed EP 4 -selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl - currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET 691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP] i , and EP 2 or EP 4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2. Copyright © 2017 the American Physiological Society.

  1. Computational active site analysis of molecular pathways to improve functional classification of enzymes.

    PubMed

    Ozyurt, A Sinem; Selby, Thomas L

    2008-07-01

    This study describes a method to computationally assess the function of homologous enzymes through small molecule binding interaction energy. Three experimentally determined X-ray structures and four enzyme models from ornithine cyclo-deaminase, alanine dehydrogenase, and mu-crystallin were used in combination with nine small molecules to derive a function score (FS) for each enzyme-model combination. While energy values varied for a single molecule-enzyme combination due to differences in the active sites, we observe that the binding energies for the entire pathway were proportional for each set of small molecules investigated. This proportionality of energies for a reaction pathway appears to be dependent on the amino acids in the active site and their direct interactions with the small molecules, which allows a function score (FS) to be calculated to assess the specificity of each enzyme. Potential of mean force (PMF) calculations were used to obtain the energies, and the resulting FS values demonstrate that a measurement of function may be obtained using differences between these PMF values. Additionally, limitations of this method are discussed based on: (a) larger substrates with significant conformational flexibility; (b) low homology enzymes; and (c) open active sites. This method should be useful in accurately predicting specificity for single enzymes that have multiple steps in their reactions and in high throughput computational methods to accurately annotate uncharacterized proteins based on active site interaction analysis. 2008 Wiley-Liss, Inc.

  2. Elucidating Oxygen Reduction Active Sites in Pyrolyzed Metal–Nitrogen Coordinated Non-Precious-Metal Electrocatalyst Systems

    PubMed Central

    2015-01-01

    Detailed understanding of the nature of the active centers in non-precious-metal-based electrocatalyst, and their role in oxygen reduction reaction (ORR) mechanistic pathways will have a profound effect on successful commercialization of emission-free energy devices such as fuel cells. Recently, using pyrolyzed model structures of iron porphyrins, we have demonstrated that a covalent integration of the Fe–Nx sites into π-conjugated carbon basal plane modifies electron donating/withdrawing capability of the carbonaceous ligand, consequently improving ORR activity. Here, we employ a combination of in situ X-ray spectroscopy and electrochemical methods to identify the various structural and functional forms of the active centers in non-heme Fe/N/C catalysts. Both methods corroboratively confirm the single site 2e– × 2e– mechanism in alkaline media on the primary Fe2+–N4 centers and the dual-site 2e– × 2e– mechanism in acid media with the significant role of the surface bound coexisting Fe/FexOy nanoparticles (NPs) as the secondary active sites. PMID:24817921

  3. Elucidating Oxygen Reduction Active Sites in Pyrolyzed Metal-Nitrogen Coordinated Non-Precious-Metal Electrocatalyst Systems.

    PubMed

    Tylus, Urszula; Jia, Qingying; Strickland, Kara; Ramaswamy, Nagappan; Serov, Alexey; Atanassov, Plamen; Mukerjee, Sanjeev

    2014-05-01

    Detailed understanding of the nature of the active centers in non-precious-metal-based electrocatalyst, and their role in oxygen reduction reaction (ORR) mechanistic pathways will have a profound effect on successful commercialization of emission-free energy devices such as fuel cells. Recently, using pyrolyzed model structures of iron porphyrins, we have demonstrated that a covalent integration of the Fe-N x sites into π-conjugated carbon basal plane modifies electron donating/withdrawing capability of the carbonaceous ligand, consequently improving ORR activity. Here, we employ a combination of in situ X-ray spectroscopy and electrochemical methods to identify the various structural and functional forms of the active centers in non-heme Fe/N/C catalysts. Both methods corroboratively confirm the single site 2e - × 2e - mechanism in alkaline media on the primary Fe 2+ -N 4 centers and the dual-site 2e - × 2e - mechanism in acid media with the significant role of the surface bound coexisting Fe/Fe x O y nanoparticles (NPs) as the secondary active sites.

  4. Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase.

    PubMed

    Ahn, Young O; Mahinthichaichan, Paween; Lee, Hyun Ju; Ouyang, Hanlin; Kaluka, Daniel; Yeh, Syun-Ru; Arjona, Davinia; Rousseau, Denis L; Tajkhorshid, Emad; Adelroth, Pia; Gennis, Robert B

    2014-10-21

    The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K(C)-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b3. The data suggest a linkage between residues lining the K(C)-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241(Vc) and the active site cross-linked Y255(Vc), as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

  5. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

    2013-07-01

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an abovegroundmore » structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)« less

  6. Recurring Slope Lineae (RSL) Observations Suggest Widespread Occurrence and Complex Behavior

    NASA Astrophysics Data System (ADS)

    Stillman, D. E.; Grimm, R. E.; Wagstaff, K.; Bue, B. D.; Michaels, T. I.

    2017-12-01

    RSL are described as narrow dark features that incrementally lengthen down steep slopes during warm seasons, fade in cold seasons, and recur annually. HiRISE observations from 5+ Mars years have allowed us to confirm 100 RSL sites and identify more than 600 candidate RSL sites. Detailed analysis of a few RSL sites has been performed using computer assisted analysis. RSL occur in low-albedo (dust-poor) regions with a latitude range of 42.2°N - 53.1°S. They are densely clustered throughout Valles Marineris (VM), in the light-toned layered deposits of Margaritifer and SW Arabia Terrae, Cerberus Fossae, and well-preserved impact craters in Chryse and Acidalia Planitae (CAP). RSL sites are also found at lower densities throughout the low-albedo highland terrains. RSL incrementally lengthen when their slopes are warm, thus the season at which RSL lengthen is dependent on latitude and slope orientation. While RSL occur on all slope orientation there is a large bias to W-facing and equatorial facing slopes. During the RSL activity season, RSL lengthening does not appear to be constant: (1) CAP RSL initially quickly lengthen and slow their lengthening rate by about an order of magnitude as temperatures increase, (2) many VM RSL sites possess RSL that fade at the same time that neighboring RSL on the same slope incrementally lengthen, and (3) some RSL sites in the southern mid-latitudes show at least two pulses of RSL activity - during the southern fall and summer RSL incrementally lengthen, fade, and then start incrementally lengthening again followed by fading as temperatures cool. The correlation of RSL activity to surface temperature, spectrally-derived hydrated salts, and quick fading all point to a wet formation mechanism. However, water sources remain problematic as water budgets suggest a much greater amount of water than could be trapped from the atmosphere. Additionally, some RSL occur in locations where subsurface discharge via an aquifer would be challenging

  7. Greenhouse Gas Fluxes at the Tablelands, NL, Canada: A Site of Active Serpentinization

    NASA Astrophysics Data System (ADS)

    Morrill, P. L.; Morrissey, L. S.; Cumming, E.

    2016-12-01

    Active sites of serpentinization have been proposed as sites for carbon capture and storage (CCS) projects. However, in addition to their ability to convert carbon dioxide to carbonate rock, sites of serpentinization also have the potential release methane, which is a more power greenhouse gas than carbon dioxide. Very little is known about the natural flux of carbon dioxide sequestered and methane released into the atmosphere from active sites of serpentinization. In this study we measured carbon dioxide, methane, and nitrous oxide gas fluxes at a pool of ultra-basic water discharging from serpentinized rock in Winterhouse Canyon, Gros Morne, Newfoundland. We found that the flux of methane released was 4.6 x 10-7 mol/m2/min and the carbon dioxide sequestered was 1.9 x 10-5 mol/m2/min, while the concentrations of nitrous oxide showed little change. Based on these fluxes we calculated predictive climate change parameters such as net radiative forcing and global warming potential which predicted that despite the methane being released the site still had an overall long-term atmospheric cooling effect based on the natural rate of carbon dioxide sequestration.

  8. Environmental controls on the activity of aquifer microbial communities in the 300 area of the Hanford site.

    PubMed

    Konopka, Allan; Plymale, Andrew E; Carvajal, Denny A; Lin, Xueju; McKinley, James P

    2013-11-01

    Aquifer microbes in the 300 Area of the Hanford Site in southeastern Washington State, USA, are located in an oligotrophic environment and are periodically exposed to U(VI) concentrations that can range up to 10 μM in small sediment fractures. Assays of (3)H-leucine incorporation indicated that both sediment-associated and planktonic microbes were metabolically active, and that organic C was growth-limiting in the sediments. Although bacteria suspended in native groundwater retained high activity when exposed to 100 μM U(VI), they were inhibited by U(VI) <1 μM in synthetic groundwater that lacked added bicarbonate. Chemical speciation modeling suggested that positively charged species and particularly (UO2)3(OH)5 (+) rose in concentration as more U(VI) was added to synthetic groundwater, but that carbonate complexes dominated U(VI) speciation in natural groundwater. U toxicity was relieved when increasing amounts of bicarbonate were added to synthetic groundwater containing 4.5 μM U(VI). Pertechnetate, an oxyanion that is another contaminant of concern at the Hanford Site, was not toxic to groundwater microbes at concentrations up to 125 μM.

  9. Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site.

    PubMed

    Wang, Lu; Fried, Stephen D; Boxer, Steven G; Markland, Thomas E

    2014-12-30

    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.

  10. Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation.

    PubMed

    Leandro, João; Stokka, Anne J; Teigen, Knut; Andersen, Ole A; Flatmark, Torgeir

    2017-07-01

    Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in l-phenylalanine (l-Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l-Phe is the key player in this transition. X-ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr 138 -loop to the active site in the presence of l-Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr 138 point mutants. A high linear correlation ( r 2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate-activated full-length tetramers. In the tetramers, a correlation ( r 2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l-Phe concentration. The new data support a similar functional importance of the Tyr 138 -loop in the catalytic domain and the full-length enzyme homotetramer.

  11. Inhibitor-based validation of a homology model of the active-site of tripeptidyl peptidase II.

    PubMed

    De Winter, Hans; Breslin, Henry; Miskowski, Tamara; Kavash, Robert; Somers, Marijke

    2005-04-01

    A homology model of the active site region of tripeptidyl peptidase II (TPP II) was constructed based on the crystal structures of four subtilisin-like templates. The resulting model was subsequently validated by judging expectations of the model versus observed activities for a broad set of prepared TPP II inhibitors. The structure-activity relationships observed for the prepared TPP II inhibitors correlated nicely with the structural details of the TPP II active site model, supporting the validity of this model and its usefulness for structure-based drug design and pharmacophore searching experiments.

  12. Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3.

    PubMed

    Shen, Hujun; Deng, Mingsen; Zhang, Yachao

    2017-10-01

    Recent crystal structures of RNA-dependent RNA polymerase (3D pol ) from Coxsackievirus B3 (CVB3) revealed that a tyrosine mutation at Phe364 (F364Y) resulted in structures with open active site whereas a hydrophobic mutation at Phe364 (F364A) led to conformations with closed active site. Besides, the crystal structures showed that the F364W mutation had no preference between the open and closed active sites, similar to wild-type. In this paper, we present a molecular dynamics (MD) study on CVB3 3D pol in order to address some important questions raised by experiments. First, MD simulations of F364Y and F364A were carried out to explore how these mutations at Phe364 influence active site dynamics and conformations. Second, MD simulations of wild-type and mutants were performed to discover the connection between active site dynamics and polymerase function. MD simulations reveal that the effect of mutations on active site dynamics is associated with the interaction between the structural motifs A and D in CVB3 3D pol . Interestingly, we discover that the active site state is influenced by the formation of a hydrogen bond between backbone atoms of Ala231 (in motif A) and Ala358 (in motif D), which has never been revealed before. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

    PubMed Central

    Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

    2005-01-01

    Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q

  14. LASIC: Light Activated Site-Specific Conjugation of Native IgGs.

    PubMed

    Hui, James Z; Tamsen, Shereen; Song, Yang; Tsourkas, Andrew

    2015-08-19

    Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large.

  15. ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity.

    PubMed

    Milgrom, Y M; Ehler, L L; Boyer, P D

    1990-11-05

    The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.

  16. c-rel activates but v-rel suppresses transcription from kappa B sites.

    PubMed Central

    Inoue, J; Kerr, L D; Ransone, L J; Bengal, E; Hunter, T; Verma, I M

    1991-01-01

    We show that the product of the protooncogene c-rel is a constituent of an NF-kappa B-like complex that binds to the kappa B site originally identified in the enhancer of immunoglobulin kappa light chain gene. c-rel protein synthesized in bacteria binds to the kappa B site in a sequence-specific manner. The rel-kappa B complex can be disrupted by incubation with anti-rel antibodies. The rel protein can form oligomers. The c-rel protein can activate transcription from promoters containing kappa B sites; v-rel, on the other hand, suppresses the transcription of genes linked to kappa B sites. Thus, v-rel may interfere with the normal transcriptional machinery of the cell by acting as a dominant negative mutant. Images PMID:2023921

  17. Omega-oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site.

    PubMed Central

    Ivanov, I; Schwarz, K; Holzhütter, H G; Myagkova, G; Kühn, H

    1998-01-01

    During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the omega-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of omega-modified fatty acids by 15-lipoxygenases and found that omega-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, omega-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403-->Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of omega-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the omega-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases. PMID:9820810

  18. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

    PubMed

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

    2016-06-15

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release.

  19. Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

    NASA Astrophysics Data System (ADS)

    Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

    1991-10-01

    Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site

  20. Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

    PubMed

    Vickers, Chris J; Olsen, Christian A; Leman, Luke J; Ghadiri, M Reza

    2012-06-14

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

  1. Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

    PubMed

    Oyama, Takahiro; Yoshimori, Atsushi; Takahashi, Satoshi; Yamamoto, Tetsuya; Sato, Akira; Kamiya, Takanori; Abe, Hideaki; Abe, Takehiko; Tanuma, Sei-Ichi

    2017-07-01

    So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Wobble pairs of the HDV ribozyme play specific roles in stabilization of active site dynamics.

    PubMed

    Sripathi, Kamali N; Banáš, Pavel; Réblová, Kamila; Šponer, Jiří; Otyepka, Michal; Walter, Nils G

    2015-02-28

    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.

  3. 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

  4. Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

    PubMed Central

    Lucas, James E; Siegel, Justin B

    2015-01-01

    Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

  5. 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.

  6. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Does your web site draw new patients?

    PubMed

    Wallin, Wendy S

    2009-11-01

    The absence of scientific data forces orthodontists to guess at how best to design Internet sites that persuade prospective patients to call for appointments. This study was conducted to identify the Web-site factors that lead prospective patients to make appointments or, conversely, to reject a practice. Ten participants actively looking online for an orthodontist were recruited to participate. They reviewed 64 orthodontic Web sites in their geographic areas and rated their likelihood of calling each practice for an appointment. The sessions were videotaped. Analysis of participant comments, navigation patterns, and ratings suggested 25 distinguishing factors. Statistical analysis showed 10 Web-site characteristics that predict the success of an orthodontic Web site in attracting new patients.

  8. Plant growth-promoting potential of bacteria isolated from active volcano sites of Barren Island, India.

    PubMed

    Amaresan, N; Kumar, K; Sureshbabu, K; Madhuri, K

    2014-02-01

    To elucidate the biodiversity of plant growth-promoting (PGP) bacteria in active volcano sites of Barren Island, India, a total of 102 bacteria were isolated and screened for their multifunctional PGP properties. The results revealed that 21 isolates (20.6%) survived heat shock at 72°C and 11 (10.8%) isolates were able to grow exposed to 25% NaCl (w/v). In assaying for PGP properties, 59 (57.8%) isolates shown indole acetic acid (IAA) like substances production, 57 isolates (55.9%) produced siderophore and 34 (33.3%) solubilized inorganic phosphate qualitatively. Whereas in the production of extracellular enzymes, 42 isolates (41.2%) produced protease and amylase, 26 (25.5%) isolates produced lipase and 24 (23.5%) isolates produced cellulase. In antagonistic activity, 30 isolates (29.4%) were found antagonistic against Macrophomina sp., 20 isolates (19.6%) against Rhizoctonia solani and 15 isolates (14.7%) against Sclerotium rolfsii. The results based on 16 rRNA gene sequencing revealed that the PGP bacteria belonged to 22 different species comprising 13 genera. Based on multifunctional properties, nine isolates were further selected to determine the PGP in brinjal and chilli seeds. Of the bacteria tested, the isolate BAN87 showed increased root and shoot length of both the crops followed in plant growth promotion by BAN86 and BAN43. The outcome of this research proves plausible practical applicability of these PGPB for crop production in soils of saline and arid environments. The present research shows diverse plant growth-promoting (PGP) bacteria could be isolated from the active volcano site and suggests that volcano sites represent an ecological niche, which harbours a diverse and hitherto largely uncharacterized microbial population with yet unknown and untapped potential biotechnological applications, for example, plant growth promoters, as evidenced from this study. The outcome of this research may have a practical effect on crop production methodologies in

  9. 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

  10. Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

    PubMed

    Lenz, Stefan A P; Wetmore, Stacey D

    2016-02-09

    Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many

  11. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

  12. 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.

  13. Evaluation of physical activity web sites for use of behavior change theories.

    PubMed

    Doshi, Amol; Patrick, Kevin; Sallis, James F; Calfas, Karen

    2003-01-01

    Physical activity (PA) Web sites were assessed for their use of behavior change theories, including constructs of the health belief model, Transtheoretical Model, social cognitive theory, and the theory of reasoned action and planned behavior. An evaluation template for assessing PA Web sites was developed, and content validity and interrater reliability were demonstrated. Two independent raters evaluated 24 PA Web sites. Web sites varied widely in application of theory-based constructs, ranging from 5 to 48 on a 100-point scale. The most common intervention strategies were general information, social support, and realistic goal areas. Coverage of theory-based strategies was low, varying from 26% for social cognitive theory to 39% for health belief model. Overall, PA Web sites provided little assessment, feedback, or individually tailored assistance for users. They were unable to substantially tailor the on-line experience for users at different stages of change or different demographic characteristics.

  14. Bear feeding activity at alpine insect aggregation sites in the Yellowstone ecosystem

    USGS Publications Warehouse

    Mattson, David J.; Gillin, Colin M.; Benson, Scott A.; Knight, Richard R.

    1991-01-01

    Bears (Ursidae) were observed from fixed-wing aircraft on or near alpine talus in the Shoshone National Forest between 15 June and 15 September in 1981–1989. Bears fed on insect aggregations at 6 known and 12 suspected alpine talus sites, disproportionately more at elevations > 3350 m, on slopes > 30°, and on south- and west-facing aspects. While at these sites, bears almost exclusively ate invertebrates, typically army cutworm moths (Euxoa auxiliaris). Subadult grizzly bears (Ursus arctos horribilis) appeared to be underrepresented at the sites, and proportionate representation of adult females with young appeared to decrease between 15 June and 15 September. Overall, observations of bears at these sites increased between 1981 and 1989. We suggest that alpine insect aggregations are an important food source for bears in the Shoshone National Forest, especially in the absence of high-quality foraging alternatives in July and August of most years.

  15. 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

    ...] Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf (OCS) Offshore... Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North Carolina... INFORMATION: Background: On December 13, 2012, BOEM published the Notice in the Federal Register (77 FR 74218...

  16. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sultatos, L.G.; Kaushik, R.

    2008-08-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, thesemore » changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.« less

  17. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew

    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 inmore » 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.« less

  18. 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.

  19. Role of mitogen-activated protein kinase (MAPK) docking sites on Staufen2 protein in dendritic mRNA transport.

    PubMed

    Nam, Yeon-Ju; Cheon, Hyo-Soon; Choi, Young-Ki; Kim, Seok-Yong; Shin, Eun-Young; Kim, Eung-Gook; Kim, Hyong Kyu

    2008-08-08

    Although transport and subsequent translation of dendritic mRNA play an important role in neuronal synaptic plasticity, the underlying mechanisms for modulating dendritic mRNA transport are almost completely unknown. In this study, we identified and characterized an interaction between Staufen2 and mitogen-activated protein kinase (MAPK) with co-immunoprecipitation assays. Staufen2 utilized a docking (D) site to interact with ERK1/2; deleting the D-site decreased colocalization of Staufen2 with immunoreactive ERK1/2 in the cell body regions of cultured hippocampal neurons, and it reduced the amount of Staufen2-containing RNP complexes in the distal dendrites. In addition, the deletion completely abolished the depolarization-induced increase of Staufen2-containing RNP complexes. These results suggest that the MAPK pathway could modulate dendritic mRNA transport through its interaction with Staufen2.

  20. Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

    PubMed Central

    Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

    2016-01-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  1. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

  2. Regional patterns of declining butternut (Juglans cinerea L.) suggest site characteristics for restoration

    Treesearch

    Randall S. Morin; Kurt W. Gottschalk; Michael E. Ostry; Andrew M. Liebhold

    2018-01-01

    Butternut trees dying from a canker disease were first reported in southwestern Wisconsin in 1967. Since then, the disease has caused extensive mortality of butternut throughout its North American range. The objectives of this study were to quantify changes in butternut populations and density across its range and identify habitat characteristics of sites where...

  3. Site clearance working group

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    The Gulf of Mexico and Louisiana continue to be areas with a high level of facility removal, and the pace of removal is projected to increase. Regulations were promulgated for the Gulf of Mexico and Louisiana requiring that abandoned sites be cleared of debris that could interfere with fishing and shrimping activities. The site clearance regulations also required verification that the sites were clear. Additionally, government programs were established to compensate fishermen for losses associated with snagging their equipment on oil and gas related objects that remained on the water bottoms in areas other than active producing sites and sitesmore » that had been verified as clear of obstructions and snags. The oil and gas industry funds the compensation programs. This paper reviews the regulations and evolving operating practices in the Gulf of Mexico and Louisiana where site clearance and fisherman`s gear compensation regulations have been in place for a number of years. Although regulations and guidelines may be in place elsewhere in the world, this paper focuses on the Gulf of Mexico and Louisiana. Workshop participants are encouraged to bring up international issues during the course of the workshop. Additionally, this paper raises questions and focuses on issues that are of concern to the various Gulf of Mexico and Louisiana water surface and water bottom stakeholders. This paper does not have answers to the questions or issues. During the workshop participants will debate the questions and issues in an attempt to develop consensus opinions and/or make suggestions that can be provided to the appropriate organizations, both private and government, for possible future research or policy adjustments. Site clearance and facility removal are different activities. Facility removal deals with removal of the structures used to produce oil and gas including platforms, wells, casing, piles, pipelines, well protection structures, etc.« less

  4. Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

    PubMed

    Bondoc, Jasper Marc G; Wolf, Nina M; Ndichuck, Michael; Abad-Zapatero, Celerino; Movahedzadeh, Farahnaz

    2017-09-01

    The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis ( Mt ), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in V max and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH - in the Thr84 residue of the wildtype of Mt FBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

  5. Active site structure and catalytic mechanism of phosphodiesterase for degradation of intracellular second messengers

    NASA Astrophysics Data System (ADS)

    Zhan, Chang-Guo

    2002-03-01

    Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulate intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it was uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand has been determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

  6. Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases*

    PubMed Central

    Nagel, Zachary D.; Cun, Shujian; Klinman, Judith P.

    2013-01-01

    A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases. PMID:23525111

  7. Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nichols,J.; Xiang, S.; Schindelin, H.

    2007-01-01

    The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe amore » detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA{sup -} crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.« less

  8. X-ray crystal structure of plasmin with tranexamic acid–derived active site inhibitors

    PubMed Central

    Wu, Guojie; Leung, Eleanor W. W.; Hidaka, Koushi; Quek, Adam J.; Caradoc-Davies, Tom T.; Jeevarajah, Devadharshini; Kirby, Nigel M.; Norton, Raymond S.; Tsuda, Yuko; Whisstock, James C.

    2017-01-01

    The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders. PMID:29296720

  9. Silver-coated nylon dressing plus active DC microcurrent for healing of autogenous skin donor sites.

    PubMed

    Malin, Edward W; Galin, Chaya M; Lairet, Kimberley F; Huzar, Todd F; Williams, James F; Renz, Evan M; Wolf, Steven E; Cancio, Leopoldo C

    2013-11-01

    Burn wounds are a significant cause of morbidity and mortality, and improved outcomes are demonstrated with early closure of both primary burn wounds and skin donor sites. Thus, technology that decreases the healing time of burns and donor sites would be potentially lifesaving. We present the results of a single-center, prospective, double-blinded, randomized controlled trial to evaluate the efficacy of silver-coated dressing with active microcurrent in comparison to silver-coated dressing with sham microcurrent on wound-closure time for autogenous skin donor sites. Four hundred five patients were screened for treatment of their donor sites using a silver-coated nylon dressing with either sham or active microcurrent stimulation. Thirty patients were enrolled in the study and then randomized. Of these, 5 patients were removed from analysis due to protocol deviations. Differences in time-to-closure were analyzed using Kaplan-Meier analysis and the proportional hazard regression model. Subjective verbal pain rating scores (0-10; 0, no pain; 10, worst pain) were also recorded. All devices were blinded and programmed at an outside facility, so that every patient had either an active or sham device. The study was unblinded only after the final patient's donor site had healed. All patients achieved donor-site healing before postoperative day 20. The 14 patients in the active microcurrent group [mean, 10.8 (2.9) days; range, 7-15 days] experienced no difference in time to wound healing as compared to the remaining patients in the sham microcurrent group [mean, 11.1 (2.0) days; range, 8-14 days; P = 0.75]. There were no differences in pain from one group compared to the other. None of the donor sites exhibited clinical signs of infection. In a sample size of 25 burn patients, the addition of direct microcurrent to silver-nylon dressings did not decrease time to wound closure of skin donor sites, and it did not show a difference in reported pain levels.

  10. STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

    PubMed

    Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

    2015-02-06

    Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the

  11. Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

    PubMed

    Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

    2014-03-07

    The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

  12. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    DOE PAGES

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; ...

    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 sitemore » 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.« less

  13. Active site-directed double mutants of dihydrofolate reductase.

    PubMed

    Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

    1996-09-15

    Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

  14. Temporal and geographic patterns of kinship structure in common dolphins (Delphinus delphis) suggest site fidelity and female-biased long-distance dispersal.

    PubMed

    Ball, Laura; Shreves, Kypher; Pilot, Małgorzata; Moura, André E

    2017-01-01

    Social structure plays a crucial role in determining a species' dispersal patterns and genetic structure. Cetaceans show a diversity of social and mating systems, but their effects on dispersal and genetic structure are not well known, in part because of technical difficulties in obtaining robust observational data. Here, we combine genetic profiling and GIS analysis to identify patterns of kin distribution over time and space, to infer mating structure and dispersal patterns in short-beaked common dolphins ( Delphinus delphis ). This species is highly social, and exhibits weak spatial genetic structure in the Northeast Atlantic and Mediterranean Sea, thought to result from fluid social structure and low levels of site fidelity. We found that although sampled groups were not composed of closely related individuals, close kin were frequently found in the same geographic location over several years. Our results suggest that common dolphin exhibits some level of site fidelity, which could be explained by foraging for temporally varying prey resource in areas familiar to individuals. Dispersal from natal area likely involves long-distance movements of females, as males are found more frequently than females in the same locations as their close kin. Long-distance dispersal may explain the near panmixia observed in this species. By analysing individuals sampled in the same geographic location over multiple years, we avoid caveats associated with divergence-based methods of inferring sex-biased dispersal. We thus provide a unique perspective on this species' social structure and dispersal behaviour, and how it relates to the observed low levels of population genetic structure in European waters. Movement patterns and social interactions are aspects of wild animal's behaviour important for understanding their ecology. However, tracking these behaviours directly can be very challenging in wide-ranging species such as whales and dolphins. In this study, we used genetic

  15. Stereoselectivity of supported alkene metathesis catalysts: a goal and a tool to characterize active sites.

    PubMed

    Copéret, Christophe

    2011-01-05

    Stereoselectivity in alkene metathesis is a challenge and can be used as a tool to study active sites under working conditions. This review describes the stereochemical relevance and problems in alkene metathesis (kinetic vs. thermodynamic issues), the use of (E/Z) ratio at low conversions as a tool to characterize active sites of heterogeneous catalysts and finally to propose strategies to improve catalysts based on the current state of the art.

  16. Interaction of mining activities and aquatic environment: A review from Greek mine sites.

    NASA Astrophysics Data System (ADS)

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

    In Greece a significant amount of mineral and ore deposits have been recorded accompanied by large industrial interest and a long mining history. Today many active and/or abandoned mine sites are scattered within the country; while mining activities take place in different sites for exploiting various deposits (clay, limestone, slate, gypsum, kaolin, mixed sulphide ores (lead, zinc, olivine, pozzolan, quartz lignite, nickel, magnesite, aluminum, bauxite, gold, marbles etc). The most prominent recent ones are: (i) the lignite exploitation that is extended in the area of Ptolemais (Western Macedonia) and Megalopolis (Central Peloponnese); and (ii) the major bauxite deposits located in central Greece within the Parnassos-Ghiona geotectonic zone and on Euboea Island. In the latter area, significant ores of magnesite were exploited and mixed sulphide ores. Centuries of intensive mining exploitation and metallurgical treatment of lead-silver deposits in Greece, have also resulted in significant abandoned sites, such as the one in Lavrion. Mining activities in Lavrio, were initiated in ancient times and continued until the 1980s, resulting in the production of significant waste stockpiles deposited in the area, crucial for the local water resources. Ιn many mining sites, environmental pressures are also recorded after the mine closure to the aquatic environment, as the surface waters flow through waste dump areas and contaminated soils. This paper aims to the geospatial visualization of the mining activities in Greece, in connection to their negative (surface- and/or ground-water pollution; overpumping due to extensive dewatering practices) or positive (enhanced groundwater recharge; pit lakes, improvement of water budget in the catchment scale) impacts on local water resources.

  17. Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access.

    PubMed

    Fritz, Timothy A; Liu, Lu; Finer-Moore, Janet S; Stroud, Robert M

    2002-06-04

    Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.

  18. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

    PubMed Central

    Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

    1992-01-01

    opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

  19. Resonance energy transfer between the active sites of creatine kinase from rabbit brain.

    PubMed

    Grossman, S H

    1990-09-03

    Resonance energy transfer was measured between the active site domains of the brain isozyme of creatine kinase (CK-BB). The reactive thiol near the active sites, one on each subunit of the dimeric protein, was derivatized using 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (AED), 2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid (AANS) and 5-iodoacetamidofluorescein (AF). Suitable donor/acceptor protein conjugated hybrids were prepared by controlled kinetics producing CK-BB-AED/AF and CK-BB-AANS/AF. Transfer efficiencies, measured from the quenching of the donor lifetime and steady-state sensitized acceptor emission, ranged from 0.10 to 0.17. From determination of the donor/acceptor overlap integrals, donor quantum yields and attempts to delimit the orientation factor using steady-state and phase-resolved anisotropy measurements, it was found that a suitable estimate of the range between the active sites was between 45 and 57 A. This range is similar to that reported previously for the muscle isozyme of creatine kinase (Grossman, S.H. (1989) Biochemistry 28, 4894-4902) but is a significantly greater distance than detected for the hybrid, myocardial specific isozyme (Grossman, S.H. (1983) Biochemistry 22, 5369-5375).

  20. Higher-order associative processing in Hermissenda suggests multiple sites of neuronal modulation.

    PubMed

    Rogers, R F; Matzel, L D

    1996-01-01

    Two important features of modern accounts of associative learning are (1) the capacity for contextual stimuli to serve as a signal for an unconditioned stimulus (US) and (2) the capacity for a previously conditioned (excitatory) stimulus to "block" learning about a redundant stimulus when both stimuli serve as a signal for the same US. Here, we examined the process of blocking, thought by some to reflect a cognitive aspect of classical conditioning, and its underlying mechanisms in the marine mollusc Hermissenda. In two behavioral experiments, a context defined by chemosensory stimuli was made excitatory by presenting unsignalled USs (rotation) in that context. The excitatory context subsequently blocked overt learning about a discrete conditioned stimulus (CS; light) paired with the US in that context. In a third experiment, the excitability of the B photoreceptors in the Hermissenda eye, which typically increases following light-rotation pairings, was examined in behaviorally blocked animals, as well as in animals that had acquired a normal CS-US association or animals that had been exposed to the CS and US unpaired. Both the behaviorally blocked and the "normal" learning groups exhibited increases in neuronal excitability relative to unpaired animals. However, light-induced multiunit activity in pedal nerves was suppressed following normal conditioning but not in blocked or unpaired control animals, suggesting that the expression of blocking is mediated by neuronal modifications not directly reflected in B-cell excitability, possibly within an extensive network of central light-responsive interneurons.

  1. An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

    PubMed

    Agnew, Christopher R J; Warrilow, Andrew G S; Burton, Nicholas M; Lamb, David C; Kelly, Steven L; Brady, R Leo

    2012-01-01

    CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 μM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

  2. An Enlarged, Adaptable Active Site in CYP164 Family P450 Enzymes, the Sole P450 in Mycobacterium leprae

    PubMed Central

    Agnew, Christopher R. J.; Warrilow, Andrew G. S.; Burton, Nicholas M.; Lamb, David C.; Kelly, Steven L.

    2012-01-01

    CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin “open” conformation of the enzyme (Kd [dissociation constant] of 0.1 μM), with binding to the low-spin “closed” form being significantly hindered (Kd of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy. PMID:22037849

  3. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    PubMed

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

  4. 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.

  5. KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

    PubMed

    Bihani, Subhash C; Chakravarty, Dhiman; Ballal, Anand

    2016-04-01

    Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes.

    PubMed

    Brown, Jenna R; Livesay, Dennis R

    2015-01-01

    β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme.

  7. Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes

    PubMed Central

    Brown, Jenna R.; Livesay, Dennis R.

    2015-01-01

    β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme. PMID:26018804

  8. The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

    PubMed Central

    Dreveny, Ingrid; Kratky, Christoph; Gruber, Karl

    2002-01-01

    The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction—the enantiospecific formation of α-hydroxynitriles—is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C–C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident. PMID:11790839

  9. Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation.

    PubMed

    Xu, Binjie; Soukup, Randal J; Jones, Christopher J; Fishel, Richard; Wozniak, Daniel J

    2016-10-01

    During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely

  10. Tauroursodeoxycholic acid binds to the G-protein site on light activated rhodopsin.

    PubMed

    Lobysheva, E; Taylor, C M; Marshall, G R; Kisselev, O G

    2018-05-01

    The heterotrimeric G-protein binding site on G-protein coupled receptors remains relatively unexplored regarding its potential as a new target of therapeutic intervention or as a secondary site of action by the existing drugs. Tauroursodeoxycholic acid bears structural resemblance to several compounds that were previously identified to specifically bind to the light-activated form of the visual receptor rhodopsin and to inhibit its activation of transducin. We show that TUDCA stabilizes the active form of rhodopsin, metarhodopsin II, and does not display the detergent-like effects of common amphiphilic compounds that share the cholesterol scaffold structure, such as deoxycholic acid. Computer docking of TUDCA to the model of light-activated rhodopsin revealed that it interacts using similar mode of binding to the C-terminal domain of transducin alpha subunit. The ring regions of TUDCA made hydrophobic contacts with loop 3 region of rhodopsin, while the tail of TUDCA is exposed to solvent. The results show that TUDCA interacts specifically with rhodopsin, which may contribute to its wide-ranging effects on retina physiology and as a potential therapeutic compound for retina degenerative diseases. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

    PubMed

    Asakura, H; Makino, S; Kobori, H; Watarai, M; Shirahata, T; Ikeda, T; Takeshi, K

    2001-08-01

    Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.

  12. Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

    PubMed Central

    Asakura, H.; Makino, S.; Kobori, H.; Watarai, M.; Shirahata, T.; Ikeda, T.; Takeshi, K.

    2001-01-01

    Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans. PMID:11561972

  13. Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

    PubMed

    Masukagami, Yumiko; Tivendale, Kelly Anne; Browning, Glenn Francis; Sansom, Fiona Margaret

    2018-02-01

    The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

  14. Trace metals from historical mining sites and past metallurgical activity remain bioavailable to wildlife today.

    PubMed

    Camizuli, Estelle; Scheifler, Renaud; Garnier, Stéphane; Monna, Fabrice; Losno, Rémi; Gourault, Claude; Hamm, Gilles; Lachiche, Caroline; Delivet, Guillaume; Chateau, Carmela; Alibert, Paul

    2018-02-21

    Throughout history, ancient human societies exploited mineral resources all over the world, even in areas that are now protected and considered to be relatively pristine. Here, we show that past mining still has an impact on wildlife in some French protected areas. We measured cadmium, copper, lead, and zinc concentrations in topsoils and wood mouse kidneys from sites located in the Cévennes and the Morvan. The maximum levels of metals in these topsoils are one or two orders of magnitude greater than their commonly reported mean values in European topsoils. The transfer to biota was effective, as the lead concentration (and to a lesser extent, cadmium) in wood mouse kidneys increased with soil concentration, unlike copper and zinc, providing direct evidence that lead emitted in the environment several centuries ago is still bioavailable to free-ranging mammals. The negative correlation between kidney lead concentration and animal body condition suggests that historical mining activity may continue to play a role in the complex relationships between trace metal pollution and body indices. Ancient mining sites could therefore be used to assess the long-term fate of trace metals in soils and the subsequent risks to human health and the environment.

  15. Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard

    Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLAmore » complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.« less

  16. 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...

  17. Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane

    DOE PAGES

    Snyder, Benjamin E. R.; Bottger, Lars H.; Bols, Max L.; ...

    2018-04-02

    Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N 2O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. As a result, density functional theory calculations clarify howmore » the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.« less

  18. Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Snyder, Benjamin E. R.; Bottger, Lars H.; Bols, Max L.

    Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N 2O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. As a result, density functional theory calculations clarify howmore » the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.« less

  19. Electric Fields at the Active Site of an Enzyme: Direct Comparison of Experiment with Theory

    NASA Astrophysics Data System (ADS)

    Suydam, Ian T.; Snow, Christopher D.; Pande, Vijay S.; Boxer, Steven G.

    2006-07-01

    The electric fields produced in folded proteins influence nearly every aspect of protein function. We present a vibrational spectroscopy technique that measures changes in electric field at a specific site of a protein as shifts in frequency (Stark shifts) of a calibrated nitrile vibration. A nitrile-containing inhibitor is used to deliver a unique probe vibration to the active site of human aldose reductase, and the response of the nitrile stretch frequency is measured for a series of mutations in the enzyme active site. These shifts yield quantitative information on electric fields that can be directly compared with electrostatics calculations. We show that extensive molecular dynamics simulations and ensemble averaging are required to reproduce the observed changes in field.

  20. The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

    PubMed

    Kee, Nalise Low Ah; Krause, Jason; Blatch, Gregory L; Muramoto, Koji; Sakka, Kazuo; Sakka, Makiko; Naudé, Ryno J; Wagner, Leona; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; Mielicki, Wojciech P; Frost, Carminita L

    2015-10-01

    Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

  1. Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

    PubMed Central

    Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

    2015-01-01

    Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

  2. Smallpox Inhibitor of Complement Enzymes (SPICE): Dissecting Functional Sites and Abrogating Activity1

    PubMed Central

    Liszewski, M. Kathryn; Leung, Marilyn K.; Hauhart, Richard; Fang, Celia J.; Bertram, Paula; Atkinson, John P.

    2010-01-01

    Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host’s immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity ~200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent. PMID:19667083

  3. Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

    PubMed

    Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

    2015-05-15

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

    DOE PAGES

    Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

    2014-12-09

    Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the

  5. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

  6. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oilmore » than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.« less

  7. The influence of the scale of mining activity and mine site remediation on the contamination legacy of historical metal mining activity.

    PubMed

    Bird, Graham

    2016-12-01

    Globally, thousands of kilometres of rivers are degraded due to the presence of elevated concentrations of potentially harmful elements (PHEs) sourced from historical metal mining activity. In many countries, the presence of contaminated water and river sediment creates a legal requirement to address such problems. Remediation of mining-associated point sources has often been focused upon improving river water quality; however, this study evaluates the contaminant legacy present within river sediments and attempts to assess the influence of the scale of mining activity and post-mining remediation upon the magnitude of PHE contamination found within contemporary river sediments. Data collected from four exemplar catchments indicates a strong relationship between the scale of historical mining, as measured by ore output, and maximum PHE enrichment factors, calculated versus environmental quality guidelines. The use of channel slope as a proxy measure for the degree of channel-floodplain coupling indicates that enrichment factors for PHEs in contemporary river sediments may also be the highest where channel-floodplain coupling is the greatest. Calculation of a metric score for mine remediation activity indicates no clear influence of the scale of remediation activity and PHE enrichment factors for river sediments. It is suggested that whilst exemplars of significant successes at improving post-remediation river water quality can be identified; river sediment quality is a much more long-lasting environmental problem. In addition, it is suggested that improvements to river sediment quality do not occur quickly or easily as a result of remediation actions focused a specific mining point sources. Data indicate that PHEs continue to be episodically dispersed through river catchments hundreds of years after the cessation of mining activity, especially during flood flows. The high PHE loads of flood sediments in mining-affected river catchments and the predicted changes to

  8. DFT study of the active site of the XoxF-type natural, cerium-dependent methanol dehydrogenase enzyme.

    PubMed

    Bogart, Justin A; Lewis, Andrew J; Schelter, Eric J

    2015-01-19

    Rare-earth metal cations have recently been demonstrated to be essential co-factors for the growth of the methanotrophic bacterium Methylacidiphilum fumariolicum SolV. A crystal structure of the rare-earth-dependent methanol dehydrogenase (MDH) includes a cerium cation in the active site. Herein, the Ce-MDH active site has been analyzed through DFT calculations. The results show the stability of the Ce(III)-pyrroloquinoline quinone (PQQ) semiquinone configuration. Calculations on the active oxidized form of this complex indicate a 0.81 eV stabilization of the PQQ(0) LUMO at cerium versus calcium, supporting the observation that the cerium cation in the active site confers a competitive advantage to Methylacidiphilum fumariolicum SolV. Using reported aqueous electrochemical data, a semi-empirical correlation was established based on cerium(IV/III) redox potentials. The correlation allowed estimation of the cerium oxidation potential of +1.35 V versus saturated calomel electrode (SCE) in the active site. The results are expected to guide the design of functional model complexes and alcohol-oxidation catalysts based on lanthanide complexes of biologically relevant quinones. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  10. Multi-Modal Active Perception for Autonomously Selecting Landing Sites on Icy Moons

    NASA Technical Reports Server (NTRS)

    Arora, A.; Furlong, P. M.; Wong, U.; Fong, T.; Sukkarieh, S.

    2017-01-01

    Selecting suitable landing sites is fundamental to achieving many mission objectives in planetary robotic lander missions. However, due to sensing limitations, landing sites which are both safe and scientifically valuable often cannot be determined reliably from orbit, particularly, in icy moon missions where orbital sensing data is noisy and incomplete. This paper presents an active perception approach to Entry Descent and Landing (EDL) which enables the lander to autonomously plan informative descent trajectories, acquire high quality sensing data during descent and exploit this additional information to select higher utility landing sites. Our approach consists of two components: probabilistic modeling of landing site features and approximate trajectory planning using a sampling based planner. The proposed framework allows the lander to plan long horizons paths and remain robust to noisy data. Results in simulated environments show large performance improvements over alternative approaches and show promise that our approach has strong potential to improve science return of not only icy moon missions but EDL systems in general.

  11. Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

    2014-09-30

    Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There aremore » correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.« less

  12. Modulation of active site electronic structure by the protein matrix to control [NiFe] hydrogenase reactivity.

    PubMed

    Smith, Dayle M A; Raugei, Simone; Squier, Thomas C

    2014-11-21

    Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni-Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

  13. Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

    PubMed

    Jiang, Xukai; Wang, Yuying; Xu, Limei; Chen, Guanjun; Wang, Lushan

    2017-09-09

    The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Unraveling the Nature of Active Sites in Ethanol Electro-oxidation by Site-Specific Marking of a Pt Catalyst with Isotope-Labeled 13CO.

    PubMed

    Farias, Manuel J S; Cheuquepán, William; Tanaka, Auro A; Feliu, Juan M

    2018-03-15

    This works deals with the identification of preferential site-specific activation at a model Pt surface during a multiproduct reaction. The (110)-type steps of a Pt(332) surface were selectively marked by attaching isotope-labeled 13 CO molecules to them, and ethanol oxidation was probed by in situ Foureir transfrom infrared spectroscopy in order to precisely determine the specific sites at which CO 2 , acetic acid, and acetaldehyde were preferentially formed. The (110) steps were active for splitting the C-C bond, but unexpectedly, we provide evidence that the pathway of CO 2 formation was preferentially activated at (111) terraces, rather than at (110) steps. Acetaldehyde was formed at (111) terraces at potentials comparable to those for CO 2 formation also at (111) terraces, while the acetic acid formation pathway became active only when the (110) steps were released by the oxidation of adsorbed 13 CO, at potentials higher than for the formation of CO 2 at (111) terraces of the stepped surface.

  15. Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways.

    PubMed

    Fischer, Carol L; Dawson, Deborah V; Blanchette, Derek R; Drake, David R; Wertz, Philip W; Brogden, Kim A

    2016-01-01

    Lipids endogenous to skin and mucosal surfaces exhibit potent antimicrobial activity against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Our previous work demonstrated the antimicrobial activity of the fatty acid sapienic acid (C(16:1Δ6)) against P. gingivalis and found that sapienic acid treatment alters both protein and lipid composition from those in controls. In this study, we further examined whole-cell protein differences between sapienic acid-treated bacteria and untreated controls, and we utilized open-source functional association and annotation programs to explore potential mechanisms for the antimicrobial activity of sapienic acid. Our analyses indicated that sapienic acid treatment induces a unique stress response in P. gingivalis resulting in differential expression of proteins involved in a variety of metabolic pathways. This network of differentially regulated proteins was enriched in protein-protein interactions (P = 2.98 × 10(-8)), including six KEGG pathways (P value ranges, 2.30 × 10(-5) to 0.05) and four Gene Ontology (GO) molecular functions (P value ranges, 0.02 to 0.04), with multiple suggestive enriched relationships in KEGG pathways and GO molecular functions. Upregulated metabolic pathways suggest increases in energy production, lipid metabolism, iron acquisition and processing, and respiration. Combined with a suggested preferential metabolism of serine, which is necessary for fatty acid biosynthesis, these data support our previous findings that the site of sapienic acid antimicrobial activity is likely at the bacterial membrane. P. gingivalis is an important opportunistic pathogen implicated in periodontitis. Affecting nearly 50% of the population, periodontitis is treatable, but the resulting damage is irreversible and eventually progresses to tooth loss. There is a great need for natural products that can be used to treat and/or prevent the overgrowth of periodontal pathogens and

  16. Determination of the Bridging Ligand in the Active Site of Tyrosinase.

    PubMed

    Zou, Congming; Huang, Wei; Zhao, Gaokun; Wan, Xiao; Hu, Xiaodong; Jin, Yan; Li, Junying; Liu, Junjun

    2017-10-28

    Tyrosinase is a type-3 copper enzyme that is widely distributed in plants, fungi, insects, and mammals. Developing high potent inhibitors against tyrosinase is of great interest in diverse fields including tobacco curing, food processing, bio-insecticides development, cosmetic development, and human healthcare-related research. In the crystal structure of Agaricus bisporus mushroom tyrosinase, there is an oxygen atom bridging the two copper ions in the active site. It is unclear whether the identity of this bridging oxygen is a water molecule or a hydroxide anion. In the present study, we theoretically determine the identity of this critical bridging oxygen by performing first-principles hybrid quantum mechanics/molecular mechanics/Poisson-Boltzmann-surface area (QM/MM-PBSA) calculations along with a thermodynamic cycle that aim to improve the accuracy. Our results show that the binding with water molecule is energy favored and the QM/MM-optimized structure is very close to the crystal structure, whereas the binding with hydroxide anions causes the increase of energy and significant structural changes of the active site, indicating that the identity of the bridging oxygen must be a water molecule rather than a hydroxide anion. The different binding behavior between water and hydroxide anions may explain why molecules with a carboxyl group or too many negative charges have lower inhibitory activity. In light of this, the design of high potent active inhibitors against tyrosinase should satisfy both the affinity to the copper ions and the charge neutrality of the entire molecule.

  17. 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

  18. An overview on GSF activities at the Semipalatinsk Test Site, Kazakhstan.

    PubMed

    Semioshkina, Natalia; Voigt, Gabrielle

    2006-02-01

    The Semipalatinsk Test Site (STS) in Kazakhstan was one of the major sites used by the former USSR for testing nuclear weapons for more than 40 years. Since the early 1990s, agricultural activities have been re-established there by neighbouring collective and private farms. Therefore, it has become important to evaluate the radiological situation and the current and future risk to people living on and using the contaminated area. During the last eight years, GSF has participated in many international projects performed on the STS to evaluate the radiological situation. A large number of soil, vegetation and food samples has been collected and analysed. Internal dose is one of the main components of the total dose when deriving risk factors for a population living within the test site. Internal doses, based on food monitoring and whole body measurements, were calculated for adults and were in the range of 13-500 microSv/y due to radiocaesium and radiostrontium.

  19. A Combined Probe-Molecule, Mössbauer, Nuclear Resonance Vibrational Spectroscopy, and Density Functional Theory Approach for Evaluation of Potential Iron Active Sites in an Oxygen Reduction Reaction Catalyst

    DOE PAGES

    Kneebone, Jared L.; Daifuku, Stephanie L.; Kehl, Jeffrey A.; ...

    2017-07-06

    While non-precious metal M-N-C (M = Fe or Co) catalysts have been developed that are effective for the oxygen reduction reaction in polymer electrolyte fuel cells, no consensus has yet been reached regarding the nature of the M sites in these heterogeneous catalysts that are responsible for reaction with dioxygen (O 2). While multiple studies have developed correlations between Fe distributions in as-prepared catalysts and ORR activity, the direct identification of sites reactive towards O 2 or O 2-analog molecules remains a significant challenge. In the present study, we demonstrate a new approach to identifying and characterizing potential Fe activemore » sites in complex ORR catalysts that combines an effective probe molecule (NO (g)) Mössbauer spectroscopy and nuclear resonance vibrational spectroscopy (NRVS) with density functional theory (DFT) calculations. Mössbauer spectroscopic studies demonstrate that NO (g) treatment of electrochemically reduced PANI-57Fe-C leads to selective reaction with only a sub-set of the Fe species present. Nuclear resonance vibrational spectroscopic studies identified new Fe-ligand vibrations associated with the site reactive towards NO (g). DFT calculations of vibrational properties of a small selection of previously proposed active site structures suggest that graphene zig-zag edge hosted Fe-N structures may be responsible for the observed vibrational behavior with NO (g) probe molecules. Moreover, such sites are likely also reactive to O 2, possibly serving as the ORR active sites in the synthesized materials.« less

  20. A Combined Probe-Molecule, Mössbauer, Nuclear Resonance Vibrational Spectroscopy, and Density Functional Theory Approach for Evaluation of Potential Iron Active Sites in an Oxygen Reduction Reaction Catalyst

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kneebone, Jared L.; Daifuku, Stephanie L.; Kehl, Jeffrey A.

    While non-precious metal M-N-C (M = Fe or Co) catalysts have been developed that are effective for the oxygen reduction reaction in polymer electrolyte fuel cells, no consensus has yet been reached regarding the nature of the M sites in these heterogeneous catalysts that are responsible for reaction with dioxygen (O 2). While multiple studies have developed correlations between Fe distributions in as-prepared catalysts and ORR activity, the direct identification of sites reactive towards O 2 or O 2-analog molecules remains a significant challenge. In the present study, we demonstrate a new approach to identifying and characterizing potential Fe activemore » sites in complex ORR catalysts that combines an effective probe molecule (NO (g)) Mössbauer spectroscopy and nuclear resonance vibrational spectroscopy (NRVS) with density functional theory (DFT) calculations. Mössbauer spectroscopic studies demonstrate that NO (g) treatment of electrochemically reduced PANI-57Fe-C leads to selective reaction with only a sub-set of the Fe species present. Nuclear resonance vibrational spectroscopic studies identified new Fe-ligand vibrations associated with the site reactive towards NO (g). DFT calculations of vibrational properties of a small selection of previously proposed active site structures suggest that graphene zig-zag edge hosted Fe-N structures may be responsible for the observed vibrational behavior with NO (g) probe molecules. Moreover, such sites are likely also reactive to O 2, possibly serving as the ORR active sites in the synthesized materials.« less

  1. New insights into the interaction between pyrrolyl diketoacids and HIV-1 integrase active site and comparison with RNase H.

    PubMed

    Corona, Angela; di Leva, Francesco Saverio; Rigogliuso, Giuseppe; Pescatori, Luca; Madia, Valentina Noemi; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Cadeddu, Marta; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; di Santo, Roberto; Tramontano, Enzo

    2016-10-01

    HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg 2+ cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding. In this study, we investigated the binding mode of four pyrrolyl DKAs to the HIV-1 IN active site by molecular modeling coupled with site-directed mutagenesis studies showing that the DKA pyrrolyl scaffold primarily interacts with the IN amino residues P145, Q146 and Q148. Importantly, the tested DKAs demonstrated good effectiveness against HIV-1 Raltegravir resistant Y143A and N155H INs, thus showing an interaction pattern with relevant differences if compared with the first generation IN inhibitors. These data provide precious insights for the design of new HIV inhibitors active on clinically selected Raltegravir resistant variants. Furthermore, this study provides new structural information to modulate IN and RNase H inhibitory activities for development of dual-acting anti-HIV agents. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

    PubMed Central

    Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

    2018-01-01

    Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity. PMID:29541639

  3. Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes.

    PubMed

    Suplatov, D A; Arzhanik, V K; Svedas, V K

    2011-01-01

    Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.

  4. 78 FR 21352 - Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-10

    ... DEPARTMENT OF ENERGY Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of Energy. ACTION: Notice of the Title X claims during fiscal... at active uranium and thorium processing sites to remediate byproduct material generated as an...

  5. Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

    PubMed Central

    Hu, L.; Borleske, B. L.; Colman, R. F.

    1997-01-01

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

  6. Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

    PubMed

    Hu, L; Borleske, B L; Colman, R F

    1997-01-01

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

  7. Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

    PubMed Central

    Huang, Weiyan; Zhao, Mei; Wei, Na; Wang, Xiaoxia; Cao, Huqing; Du, Quan; Liang, Zicai

    2014-01-01

    Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10−5 mg/ml- 10−1 mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types. PMID:24805924

  8. The active site of O-GlcNAc transferase imposes constraints on substrate sequence

    PubMed Central

    Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

    2016-01-01

    O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

  9. Activation of both acfA and acfD transcription by Vibrio cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat conformation.

    PubMed

    Withey, Jeffrey H; DiRita, Victor J

    2005-05-01

    The Gram-negative bacterium Vibrio cholerae is the infectious agent responsible for the disease Asiatic cholera. The genes required for V. cholerae virulence, such as those encoding the cholera toxin (CT) and toxin-coregulated pilus (TCP), are controlled by a cascade of transcriptional activators. Ultimately, the direct transcriptional activator of the majority of V. cholerae virulence genes is the AraC/XylS family member ToxT protein, the expression of which is activated by the ToxR and TcpP proteins. Previous studies have identified the DNA sites to which ToxT binds upstream of the ctx operon, encoding CT, and the tcpA operon, encoding, among other products, the major subunit of the TCP. These known ToxT binding sites are seemingly dissimilar in sequence other than being A/T rich. Further results suggested that ctx and tcpA each has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptional lacZ fusions and in vitro copper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribed acfA and acfD genes, which encode components of the accessory colonization factor required for efficient intestinal colonization by V. cholerae. Our results indicate that ToxT binds to a pair of DNA sites between acfA and acfD in an inverted repeat orientation. Moreover, a mutational analysis of the ToxT binding sites indicates that both binding sites are required by ToxT for transcriptional activation of both acfA and acfD. Using copper-phenanthroline footprinting to assess the occupancy of ToxT on DNA having mutations in one of these binding sites, we found that protection by ToxT of the unaltered binding site was not affected, whereas protection by ToxT of the mutant binding site was significantly reduced in the region of the mutations. The results of further footprinting experiments using DNA templates having +5 bp and +10 bp

  10. Retrieving atmospheric transmissivity for biologically active daily dose, in various european sites

    NASA Astrophysics Data System (ADS)

    de La Casinière, A.; Touré, M. L.; Lenoble, J.; Cabot, T.

    2003-04-01

    In the frame of the European Project EDUCE, global UV irradiance spectra recorded all along the year in several European sites are stored in a common database located in Finland. From the spectra set of some of these stations, are calculated atmospheric transmissivities for daily doses of four biologically active UV radiation, namely: UV-B, erythema, DNA damage, and plant damage. A transmissivity is defined as the ratio of the ground level value of the daily dose of interest to its corresponding extra-atmospheric value. Multiple linear correlation of the various transmissivities with three predictors (daily sunshine fraction, cosine of the daily minimum SZA, and daily total ozone column) assumed to be independent variables, are done for year 2000. The coefficients obtained from year 2000 correlation in a given site are expected to retrieve, from the local predictors, the daily dose for year 2001 in the same site, the average error being lesser than 10% for monthly mean values, and lesser than 5% for three-monthly mean values, depending on the daily dose type. Comparison of yearly mean daily doses retrieved in a given site from coefficients obtained in other sites is also presented.

  11. Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

    PubMed

    Chung, Ivy Yeuk Wah; Paetzel, Mark

    2013-05-03

    Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

  12. An additional substrate binding site in a bacterial phenylalanine hydroxylase

    PubMed Central

    Ronau, Judith A.; Paul, Lake N.; Fuchs, Julian E.; Corn, Isaac R.; Wagner, Kyle T.; Liedl, Klaus R.; Abu-Omar, Mahdi M.; Das, Chittaranjan

    2014-01-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

  13. Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

    PubMed Central

    Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

    2015-01-01

    Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins. PMID:26582921

  14. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard

    2004-12-20

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associatedmore » with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.« less

  15. Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine

    PubMed Central

    Li, Bin; Schopfer, Lawrence M.; Grigoryan, Hasmik; Thompson, Charles M.; Hinrichs, Steven H.; Masson, Patrick; Lockridge, Oksana

    2009-01-01

    The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation. PMID:18930948

  16. Approaches to efficient molecular catalyst systems for photochemical H2 production using [FeFe]-hydrogenase active site mimics.

    PubMed

    Wang, Mei; Chen, Lin; Li, Xueqiang; Sun, Licheng

    2011-12-28

    The research on structural and functional biomimics of the active site of [FeFe]-hydrogenases is in an attempt to elucidate the mechanisms of H(2)-evolution and uptake at the [FeFe]-hydrogenase active site, and to learn from Nature how to create highly efficient H(2)-production catalyst systems. Undoubtedly, it is a challenging, arduous, and long-term work. In this perspective, the progresses in approaches to photochemical H(2) production using mimics of the [FeFe]-hydrogenase active site as catalysts in the last three years are reviewed, with emphasis on adjustment of the redox potentials and hydrophilicity of the [FeFe]-hydrogenase active site mimics to make them efficient catalysts for H(2) production. With gradually increasing understanding of the chemistry of the [FeFe]-hydrogenases and their mimics, more bio-inspired proton reduction catalysts with significantly improved efficiency of H(2) production will be realized in the future. This journal is © The Royal Society of Chemistry 2011

  17. Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

    PubMed Central

    Qiao, Huan; May, James M.

    2012-01-01

    Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC) region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a “bridge” mechanism with upstream sequences. PMID:22532872

  18. The influence of small-mammal burrowing activity on water storage at the Hanford Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Landeen, D.S.

    This paper summarizes the activities that were conducted in support of the long-term surface barrier development program by Westinghouse Hanford Company to determine the degree that small-mammal burrow systems affect the loss or retention of water in the soils at the Hanford Site in Washington state. An animal intrusion lysimeter facility was constructed, consisting of two outer boxes buried at grade, which served as receptacles for six animal intrusion lysimeters. Small burrowing animals common the Hanford Site were introduced over a 3- to 4-month period. Supplemental precipitation was added monthly to three of the lysimeters with a rainfall simulator (rainulator).more » Information collected from the five tests indicated that (1) during summer months, water was lost in all the lysimeters, including the supplemental precipitation added with the rainulator; and (2) during winter months, all lysimeters gained water. The data indicate little difference in the amount of water stored between control and animal lysimeters. The overall water loss was attributed to surface evaporation, a process that occurred equally in control and treatment lysimeters. Other causes of water loss are a result of (1) constant soil turnover and subsequent drying, and (2) burrow ventilation effects. This suggests that burrow systems will not contribute to any significant water storage at depth and, in fact, may enhance the removal of water from the soil.« less

  19. “Gate-keeper” Residues and Active-Site Rearrangements in DNA Polymerase μ Help Discriminate Non-cognate Nucleotides

    PubMed Central

    Li, Yunlang; Schlick, Tamar

    2013-01-01

    Incorporating the cognate instead of non-cognate substrates is crucial for DNA polymerase function. Here we analyze molecular dynamics simulations of DNA polymerase μ (pol μ) bound to different non-cognate incoming nucleotides including A:dCTP, A:dGTP, A(syn):dGTP, A:dATP, A(syn):dATP, T:dCTP, and T:dGTP to study the structure-function relationships involved with aberrant base pairs in the conformational pathway; while a pol μ complex with the A:dTTP base pair is available, no solved non-cognate structures are available. We observe distinct differences of the non-cognate systems compared to the cognate system. Specifically, the motions of active-site residue His329 and Asp330 distort the active site, and Trp436, Gln440, Glu443 and Arg444 tend to tighten the nucleotide-binding pocket when non-cognate nucleotides are bound; the latter effect may further lead to an altered electrostatic potential within the active site. That most of these “gate-keeper” residues are located farther apart from the upstream primer in pol μ, compared to other X family members, also suggests an interesting relation to pol μ's ability to incorporate nucleotides when the upstream primer is not paired. By examining the correlated motions within pol μ complexes, we also observe different patterns of correlations between non-cognate systems and the cognate system, especially decreased interactions between the incoming nucleotides and the nucleotide-binding pocket. Altered correlated motions in non-cognate systems agree with our recently proposed hybrid conformational selection/induced-fit models. Taken together, our studies propose the following order for difficulty of non-cognate system insertions by pol μ: T:dGTP

  20. Spatially-Explicit Assessments of Genetic Biodiversity and Dispersal in Gopher Tortoises for Evaluation of Habitat Fragmentation at DoD Sites

    DTIC Science & Technology

    2008-10-01

    tortoise refuge on Camp Shelby). The markers showed that the no military activity sites they are closer to the high- impact sites than they are to...19 km south of Hattiesburg. The facility contains a 15 km2 “ impact area” used for artillery training activities. Activity within this area includes...However, these sites are more closely related to the impacted sites (based upon patterns of clustering) than that are to each other. This suggests

  1. Bacterial Abundance and Activity across Sites within Two Northern Wisconsin Sphagnum Bogs.

    PubMed

    Fisher; Graham; Graham

    1998-11-01

    Abstract Bacterial abundance, temperature, pH, and dissolved organic carbon (DOC) concentration were compared across surface sites within and between two northern Wisconsin Sphagnum peatlands over the summer seasons in 1995 and 1996. Sites of interest were the Sphagnum mat surface, the water-filled moat (lagg) at the bog margin, and the bog lake littoral zone. Significant differences in both bacterial populations and water chemistry were observed between sites. pH was highest in the lake and lowest in the mat at both bogs; the opposite was true for DOC. Large populations of bacteria were present in surface interstitial water from the mat; abundance in this site was consistently higher than in the moat or lake. Bacterial abundance also increased across sites of increasing DOC concentration and declining pH. Bacterial activities (rates of [3H]leucine incorporation) and growth in dilution cultures (with grazers removed) were also assessed in lake, moat, and mat sites. Results using these measures generally supported the trends observed in abundance, although high rates of [3H]leucine incorporation were recorded in the moat at one of the bogs. Our results indicate that bacterial populations in Sphagnum peatlands are not adversely affected by acidity, and that DOC may be more important than pH in determining bacterial abundance in these environments.

  2. Computational Investigations of Trichoderma Reesei Cel7A Suggest New Routes for Enzyme Activity Improvements

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beckham, G. T.; Payne, C. M.; Bu, L.

    2012-01-01

    The Trichoderma reesei Family 7 cellulase (Cel7A) is a key industrial enzyme in the production of biofuels from lignocellulosic biomass. It is a multi-modular enzyme with a Family 1 carbohydrate-binding module, a flexible O-glycosylated linker, and a large catalytic domain. We have used simulation to elucidate new functions for the 3 sub-domains, which suggests new routes to increase the activity of this central enzyme. These findings include new roles for glycosylation, which we have shown can be used to tune the binding affinity. We have also examined the structures of the catalytically-active complex of Cel7A and its non-processive counterpart, Cel7B,more » engaged on cellulose, which suggests allosteric mechanisms involved in chain binding when these cellulases are complexed on cellulose. Our computational results also suggest that product inhibition varies significantly between Cel7A and Cel7B, and we offer a molecular-level explanation for this observation. Finally, we discuss simulations of the absolute and relative binding free energy of cellulose ligands and various mutations along the CD tunnel, which will affect processivity and the ability of Cel7A (and related enzymes) to digest cellulose. These results highlight new considerations in protein engineering for processive and non-processive cellulases for production of lignocellulosic biofuels.« less

  3. Design, synthesis and biological activity of novel donepezil derivatives bearing N-benzyl pyridinium moiety as potent and dual binding site acetylcholinesterase inhibitors.

    PubMed

    Lan, Jin-Shuai; Zhang, Tong; Liu, Yun; Yang, Jing; Xie, Sai-Sai; Liu, Jing; Miao, Ze-Yang; Ding, Yue

    2017-06-16

    A series of new donepezil derivatives were designed synthesized and evaluated as multifunctional cholinesterase inhibitors against Alzheimer's disease (AD). In vitro studies showed that most of them exhibited significant potency to inhibit acetylcholinesterase and self-induced β-amyloid (Aβ) aggregation, and moderate antioxidant activity. Especially, compound 5b presented the greatest ability to inhibit cholinesterase (IC 50 , 1.9 nM for eeAChE and 0.8 nM for hAChE), good inhibition of Aβ aggregation (53.7% at 20 μM) and good antioxidant activity (0.54 trolox equivalents). Kinetic and molecular modeling studies indicated that compound 5b was a mixed-type inhibitor, binding simultaneously to the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, compound 5b could reduce PC12 cells death induced by oxidative stress and Aβ (1-42). Moreover, in vivo experiments showed that compound 5b was nontoxic and tolerated at doses up to 2000 mg/kg. These results suggested that compound 5b might be an excellent multifunctional agent for AD treatment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Spectroscopic insights into the nature of active sites in iron–nitrogen–carbon electrocatalysts for oxygen reduction in acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jia, Qingying; Ramaswamy, Nagappan; Tylus, Urszula

    Developing efficient and inexpensive catalysts for the sluggish oxygen reduction reaction (ORR) constitutes one of the grand challenges in the fabrication of commercially viable fuel cell devices and metal–air batteries for future energy applications. Despite recent achievements in designing advanced Pt-based and Pt-free catalysts, current progress primarily involves an empirical approach of trial-and-error combination of precursors and synthesis conditions, which limits further progress. Rational design of catalyst materials requires proper understanding of the mechanistic origin of the ORR and the underlying surface properties under operating conditions that govern catalytic activity. Herein, several different groups of iron-based catalysts synthesized via differentmore » methods and/or precursors were systematically studied by combining multiple spectroscopic techniques under ex situ and in situ conditions in an effort to obtain a comprehensive understanding of the synthesis-products correlations, nature of active sites, and the reaction mechanisms. These catalysts include original macrocycles, macrocycle-pyrolyzed catalysts, and Fe-N–C catalysts synthesized from individual Fe, N, and C precursors including polymer-based catalysts, metal organic framework (MOF)-based catalysts, and sacrificial support method (SSM)-based catalysts. The latter group of catalysts is most promising as not only they exhibit exceptional ORR activity and/or durability, but also the final products are controllable. We show that the high activity observed for most pyrolyzed Fe-based catalysts can mainly be attributed to a single active site: non-planar Fe–N 4 moiety embedded in distorted carbon matrix characterized by a high potential for the Fe 2+/3+ redox transition in acidic electrolyte/environment. The high intrinsic ORR activity, or turnover frequency (TOF), of this site is shown to be accounted for by redox catalysis mechanism that highlights the dominant role of the site

  5. LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

    PubMed

    Peterson, Kenneth R; Fedosyuk, Halyna; Harju-Baker, Susanna

    2012-12-01

    The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c). The 5'ΔHS3c mice expressed β(m)-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5'HS3 core was not required for β(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5'HS complete deletion mice, except β(m)-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.

  6. An alternate binding site for PPARγ ligands

    PubMed Central

    Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

    2014-01-01

    PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

  7. Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

    PubMed

    Siqueira, Andrei Santos; Lima, Alex Ranieri Jerônimo; Dall'Agnol, Leonardo Teixeira; de Azevedo, Juliana Simão Nina; da Silva Gonçalves Vianez, João Lídio; Gonçalves, Evonnildo Costa

    2016-03-01

    Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

  8. Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

    PubMed

    Mohr, S; Stamler, J S; Brüne, B

    1994-07-18

    Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

  9. Real-Time Visualization of Active Species in a Single-Site Metal–Organic Framework Photocatalyst

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Sizhuo; Pattengale, Brian; Lee, Sungsik

    In this work, we report a new single-site photocatalyst (Co-Ru-UIO- 67(bpy)) based on a metal-organic framework platform with incorporated molecular photosensitizer and catalyst. We show that this catalyst not only demonstrates exceptional activity for light-driven H2 production but also can be recycled without loss of activity. Using the combination of optical transient absorption spectroscopy and in situ X-ray absorption spectroscopy, we not only captured the key CoI intermediate species formed after ultrafast charge transfer from the incorporated photosensitizer but also identified the rate-limiting step in the catalytic cycle, providing insight into the catalysis mechanism of these single-site metal-organic framework photocatalysts.

  10. COMPARISON OF THE EXTENT OF TREATMENT OF MTBE AND BENZENE BY ACTIVE REMEDIAL TECHNOLOGY AT UST SITES IN KANSAS

    EPA Science Inventory

    Data were collected from 63 sites in Kansas where technology for active cleanup of gasoline contamination had been implemented; SVE and AS was used at 39 sites, SVE alone at 11 sites, SVE an AS and excavation at 6 sites, SVE and product recovery at 3 sites, excavation alone at 2 ...

  11. CNS sites activated by renal pelvic epithelial sodium channels (ENaCs) in response to hypertonic saline in awake rats.

    PubMed

    Goodwill, Vanessa S; Terrill, Christopher; Hopewood, Ian; Loewy, Arthur D; Knuepfer, Mark M

    2017-05-01

    In some patients, renal nerve denervation has been reported to be an effective treatment for essential hypertension. Considerable evidence suggests that afferent renal nerves (ARN) and sodium balance play important roles in the development and maintenance of high blood pressure. ARN are sensitive to sodium concentrations in the renal pelvis. To better understand the role of ARN, we infused isotonic or hypertonic NaCl (308 or 500mOsm) into the left renal pelvis of conscious rats for two 2hours while recording arterial pressure and heart rate. Subsequently, brain tissue was analyzed for immunohistochemical detection of the protein Fos, a marker for neuronal activation. Fos-immunoreactive neurons were identified in numerous sites in the forebrain and brainstem. These areas included the nucleus tractus solitarius (NTS), the lateral parabrachial nucleus, the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). The most effective stimulus was 500mOsm NaCl. Activation of these sites was attenuated or prevented by administration of benzamil (1μM) or amiloride (10μM) into the renal pelvis concomitantly with hypertonic saline. In anesthetized rats, infusion of hypertonic saline but not isotonic saline into the renal pelvis elevated ARN activity and this increase was attenuated by simultaneous infusion of benzamil or amiloride. We propose that renal pelvic epithelial sodium channels (ENaCs) play a role in activation of ARN and, via central visceral afferent circuits, this system modulates fluid volume and peripheral blood pressure. These pathways may contribute to the development of hypertension. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

    PubMed Central

    Batt, C A; Jamieson, A C; Vandeyar, M A

    1990-01-01

    Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme. Images PMID:2405386

  13. The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases*

    PubMed Central

    Peterson, Ryan L.; Galaleldeen, Ahmad; Villarreal, Johanna; Taylor, Alexander B.; Cabelli, Diane E.; Hart, P. John; Culotta, Valeria C.

    2016-01-01

    In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or “pseudofungi” species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies. PMID:27535222

  14. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Dynamics of the active site architecture in plant-type ferredoxin-NADP(+) reductases catalytic complexes.

    PubMed

    Sánchez-Azqueta, Ana; Catalano-Dupuy, Daniela L; López-Rivero, Arleth; Tondo, María Laura; Orellano, Elena G; Ceccarelli, Eduardo A; Medina, Milagros

    2014-10-01

    Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Skinfold thickness at 8 common cryotherapy sites in various athletic populations.

    PubMed

    Jutte, Lisa S; Hawkins, Jeremy; Miller, Kevin C; Long, Blaine C; Knight, Kenneth L

    2012-01-01

    Researchers have observed slower cooling rates in thigh muscle with greater overlying adipose tissue, suggesting that cryotherapy duration should be based on the adipose thickness of the treatment site. Skinfold data do not exist for other common cryotherapy sites, and no one has reported how those skinfolds might vary because of physical activity level or sex. To determine the variability in skinfold thickness among common cryotherapy sites relative to sex and activity level (National Collegiate Athletic Association Division I athletes, recreationally active college athletes). Descriptive laboratory study. Field. Three hundred eighty-nine college students participated; 196 Division I athletes (157 men, 39 women) were recruited during preseason physicals, and 193 recreationally active college athletes (108 men, 85 women) were recruited from physical education classes. Three skinfold measurements to within 1 mm were taken at 8 sites (inferior angle of the scapula, middle deltoid, ulnar groove, midforearm, midthigh, medial collateral ligament, midcalf, and anterior talofibular ligament [ATF]) using Lange skinfold calipers. Skinfold thickness in millimeters. We noted interactions among sex, activity level, and skinfold site. Male athletes had smaller skinfold measurements than female athletes at all sites except the ATF, scapula, and ulnar groove (F₇,₂₇₀₂ = 69.85, P < .001). Skinfold measurements were greater for recreationally active athletes than their Division I counterparts at all sites except the ATF, deltoid, and ulnar groove (F₇,₂₇₀₂ = 30.79, P < .001). Thigh skinfold measurements of recreationally active female athletes were the largest, and their ATF skinfolds were the smallest. Skinfold thickness at common cryotherapy treatment sites varied based on level of physical activity and sex. Therefore, clinicians should measure skinfold thickness to determine an appropriate cryotherapy duration.

  17. Skinfold Thickness at 8 Common Cryotherapy Sites in Various Athletic Populations

    PubMed Central

    Jutte, Lisa S.; Hawkins, Jeremy; Miller, Kevin C.; Long, Blaine C.; Knight, Kenneth L.

    2012-01-01

    Context: Researchers have observed slower cooling rates in thigh muscle with greater overlying adipose tissue, suggesting that cryotherapy duration should be based on the adipose thickness of the treatment site. Skinfold data do not exist for other common cryotherapy sites, and no one has reported how those skinfolds might vary because of physical activity level or sex. Objective: To determine the variability in skinfold thickness among common cryotherapy sites relative to sex and activity level (National Collegiate Athletic Association Division I athletes, recreationally active college athletes). Design: Descriptive laboratory study. Setting: Field. Patients or Other Participants: Three hundred eighty-nine college students participated; 196 Division I athletes (157 men, 39 women) were recruited during preseason physicals, and 193 recreationally active college athletes (108 men, 85 women) were recruited from physical education classes. Intervention(s): Three skinfold measurements to within 1 mm were taken at 8 sites (inferior angle of the scapula, middle deltoid, ulnar groove, midforearm, midthigh, medial collateral ligament, midcalf, and anterior talofibular ligament [ATF]) using Lange skinfold calipers. Main Outcome Measure(s): Skinfold thickness in millimeters. Results: We noted interactions among sex, activity level, and skinfold site. Male athletes had smaller skinfold measurements than female athletes at all sites except the ATF, scapula, and ulnar groove (F7,2702 = 69.85, P < .001). Skinfold measurements were greater for recreationally active athletes than their Division I counterparts at all sites except the ATF, deltoid, and ulnar groove (F7,2702 = 30.79, P < .001). Thigh skinfold measurements of recreationally active female athletes were the largest, and their ATF skinfolds were the smallest. Conclusions: Skinfold thickness at common cryotherapy treatment sites varied based on level of physical activity and sex. Therefore, clinicians should measure

  18. Ni-Nanocluster Modified Black TiO2 with Dual Active Sites for Selective Photocatalytic CO2 Reduction.

    PubMed

    Billo, Tadesse; Fu, Fang-Yu; Raghunath, Putikam; Shown, Indrajit; Chen, Wei-Fu; Lien, Hsiang-Ting; Shen, Tzu-Hsien; Lee, Jyh-Fu; Chan, Ting-Shan; Huang, Kuo-You; Wu, Chih-I; Lin, M C; Hwang, Jih-Shang; Lee, Chih-Hao; Chen, Li-Chyong; Chen, Kuei-Hsien

    2018-01-01

    One of the key challenges in artificial photosynthesis is to design a photocatalyst that can bind and activate the CO 2 molecule with the smallest possible activation energy and produce selective hydrocarbon products. In this contribution, a combined experimental and computational study on Ni-nanocluster loaded black TiO 2 (Ni/TiO 2[Vo] ) with built-in dual active sites for selective photocatalytic CO 2 conversion is reported. The findings reveal that the synergistic effects of deliberately induced Ni nanoclusters and oxygen vacancies provide (1) energetically stable CO 2 binding sites with the lowest activation energy (0.08 eV), (2) highly reactive sites, (3) a fast electron transfer pathway, and (4) enhanced light harvesting by lowering the bandgap. The Ni/TiO 2[Vo] photocatalyst has demonstrated highly selective and enhanced photocatalytic activity of more than 18 times higher solar fuel production than the commercial TiO 2 (P-25). An insight into the mechanisms of interfacial charge transfer and product formation is explored. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Incorporating ToxCast and Tox21 datasets to rank biological activity of chemicals at Superfund sites in North Carolina.

    PubMed

    Tilley, Sloane K; Reif, David M; Fry, Rebecca C

    2017-04-01

    The Superfund program of the Environmental Protection Agency (EPA) was established in 1980 to address public health concerns posed by toxic substances released into the environment in the United States. Forty-two of the 1328 hazardous waste sites that remain on the Superfund National Priority List are located in the state of North Carolina. We set out to develop a database that contained information on both the prevalence and biological activity of chemicals present at Superfund sites in North Carolina. A chemical characterization tool, the Toxicological Priority Index (ToxPi), was used to rank the biological activity of these chemicals based on their predicted bioavailability, documented associations with biological pathways, and activity in in vitro assays of the ToxCast and Tox21 programs. The ten most prevalent chemicals found at North Carolina Superfund sites were chromium, trichloroethene, lead, tetrachloroethene, arsenic, benzene, manganese, 1,2-dichloroethane, nickel, and barium. For all chemicals found at North Carolina Superfund sites, ToxPi analysis was used to rank their biological activity. Through this data integration, residual pesticides and organic solvents were identified to be some of the most highly-ranking predicted bioactive chemicals. This study provides a novel methodology for creating state or regional databases of biological activity of contaminants at Superfund sites. These data represent a novel integrated profile of the most prevalent chemicals at North Carolina Superfund sites. This information, and the associated methodology, is useful to toxicologists, risk assessors, and the communities living in close proximity to these sites. Copyright © 2016. Published by Elsevier Ltd.

  20. Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site.

    PubMed

    Shukla, Rohit; Shukla, Harish; Tripathi, Timir

    2018-01-01

    Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Site fidelity and condition metrics suggest sequential habitat use by early juvenile snook

    USGS Publications Warehouse

    Brame, Adam B.; McIvor, Carole; Peebles, Ernst B; Hollander, David J.

    2014-01-01

    The common snook Centropomus undecimalis is an estuarine-dependent fish that relies on landward wetlands as nursery habitat. Despite its economic importance, portions of the snook's early life history are poorly understood. We compared habitat use of young-of-the-year (YOY) snook in 2 geomorphic mesohabitats (tidal pond and tidal creek) along an estuarine gradient (upstream vs. downstream) within a single wetland during fall recruitment. We used abundance, length, condition indices, and stable isotopes to assess ontogenetic mesohabitat use and site fidelity. We found that (1) YOY snook were more abundant within the upstream creek and ponds; (2) the smallest snook were found only in ponds; (3) snook from ponds had lower condition (Fulton's K and hepatosomatic index); (4) snook began moving from ponds to the creek at ~40 mm standard length; and (5) snook from the 2 mesohabitats were isotopically distinct, indicating high site fidelity at rather small spatial scales. Collectively, these data identified sequential use of mesohabitats, wherein seaward-spawned YOY snook moved landward and recruited to pond habitats, where they dedicated energy to growth (as length) before making an ontogenetic habitat shift to the creek. Once in the creek, YOY snook condition improved as they approached maturity and started the downstream return towards seaward locations. The wetland network that was previously viewed as generalized nursery habitat instead consists of mesohabitats that support different life stages in sequence. This represents ontogenetic habitat complementation, in which lower availability of a required mesohabitat type may limit the entire wetland's contribution to the adult population.

  2. Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

    PubMed Central

    Jayasena, S D; Johnston, B H

    1992-01-01

    tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

  3. Is There a Role for Social Networking Sites in Education?

    NASA Astrophysics Data System (ADS)

    Santos, Ieda M.; Hammond, Michael; Durli, Zenilde; Chou, Shiao-Yuh

    Social networking sites such as Facebook and MySpace have become popular among millions of users including students of all ages. There are ongoing discussions over the potential of these sites to support teaching and learning, particularly to complement traditional or online classroom activities. This paper explores whether social networking have a place in teaching and learning by investigating how students use these sites and whether they find opportunities to discuss study related activities with their peers. Two small scale studies were carried out in a face-to-face undergraduate course in Singapore and students enrolled in a face-to-face Master’s programme in Brazil. Data were collected using surveys and interviews; findings were mixed. Many of the Brazilian students used social networking sites to both socialize and discuss their studies while the Singaporean students used such sites for social interactions only. The paper discusses these differences and offers suggestions for further research.

  4. Association between Latest Activated Sites in the Left Ventricle and Akinetic Segments in Patients with Ischemic Cardiomyopathy.

    PubMed

    Sadeghian, Hakimeh; Kousari, Aliasghar; Majidi, Shahla; Rezvanfard, Mehrnaz; Kazemisaeid, Ali; Moezzi, Seyed Ali; Vasheghani Farahani, Ali; Abdar Esfahani, Morteza; Sahebjam, Mohammad; Zoroufian, Arezoo; Sadeghian, Afsaneh

    2016-07-06

    Background: It is not clear whether the latest activation sites in the left ventricle (LV) are matched with infracted regions in patients with ischemic cardiomyopathy (ICM). We aimed to investigate whether the latest activation sites in the LV are in agreement with the region of akinesia in patients with ICM. Methods: Data were analyzed in 106 patients (age = 60.5 ± 12.1 y, male = 88.7%) with ICM (ejection fraction ≤ 35%) who were refractory to pharmacological therapy and were referred to the echocardiography department for an evaluation of the feasibility of cardiac resynchronization therapy. Wall motion abnormalities, time to peak systolic myocardial velocity (Ts) of 6 basal and 6 mid-portion segments of the LV, and 4 frequently used dyssynchrony indices were measured using 2-dimensional echocardiography and tissue Doppler imaging (TDI). To evaluate the influence of the electrocardiographic pattern, we categorized the patients into 2 groups: patients with QRS ≤ 120 ms and those with QRS >120 ms. Results: A total of 1 272 segments were studied. The latest activation sites (with longest Ts) were most frequently located in the mid-anterior (n = 32, 30.2%) and basal-anterior segments (n = 29, 27.4%), while the most common sites of akinesia were the mid-anteroseptal (n = 65, 61.3%) and mid-septal (n = 51, 48.1%) segments. Generally, no significant concordance was found between the latest activated segments and akinesia either in all the patients or in the QRS groups. Detailed analysis within the segments indicated a good agreement between akinesia and delayed activation in the basal-lateral segment solely in the patients with QRS duration ≤ 120 ms (Φ = 0.707; p value ≤ 0.001). Conclusion: The akinetic segment on 2-dimensional echocardiogram was not matched with the latest activation sites in the LV determined by TDI in patients with ICM.

  5. Association between Latest Activated Sites in the Left Ventricle and Akinetic Segments in Patients with Ischemic Cardiomyopathy

    PubMed Central

    Sadeghian, Hakimeh; Kousari, Aliasghar; Majidi, Shahla; Rezvanfard, Mehrnaz; Kazemisaeid, Ali; Moezzi, Seyed Ali; Vasheghani Farahani, Ali; Abdar Esfahani, Morteza; Sahebjam, Mohammad; Zoroufian, Arezoo; Sadeghian, Afsaneh

    2016-01-01

    Background: It is not clear whether the latest activation sites in the left ventricle (LV) are matched with infracted regions in patients with ischemic cardiomyopathy (ICM). We aimed to investigate whether the latest activation sites in the LV are in agreement with the region of akinesia in patients with ICM. Methods: Data were analyzed in 106 patients (age = 60.5 ± 12.1 y, male = 88.7%) with ICM (ejection fraction ≤ 35%) who were refractory to pharmacological therapy and were referred to the echocardiography department for an evaluation of the feasibility of cardiac resynchronization therapy. Wall motion abnormalities, time to peak systolic myocardial velocity (Ts) of 6 basal and 6 mid-portion segments of the LV, and 4 frequently used dyssynchrony indices were measured using 2-dimensional echocardiography and tissue Doppler imaging (TDI). To evaluate the influence of the electrocardiographic pattern, we categorized the patients into 2 groups: patients with QRS ≤ 120 ms and those with QRS >120 ms. Results: A total of 1 272 segments were studied. The latest activation sites (with longest Ts) were most frequently located in the mid-anterior (n = 32, 30.2%) and basal-anterior segments (n = 29, 27.4%), while the most common sites of akinesia were the mid-anteroseptal (n = 65, 61.3%) and mid-septal (n = 51, 48.1%) segments. Generally, no significant concordance was found between the latest activated segments and akinesia either in all the patients or in the QRS groups. Detailed analysis within the segments indicated a good agreement between akinesia and delayed activation in the basal-lateral segment solely in the patients with QRS duration ≤ 120 ms (Φ = 0.707; p value ≤ 0.001). Conclusion: The akinetic segment on 2-dimensional echocardiogram was not matched with the latest activation sites in the LV determined by TDI in patients with ICM. PMID:27956911

  6. The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site.

    PubMed

    Mizanur, Rahman M; Frasca, Verna; Swaminathan, Subramanyam; Bavari, Sina; Webb, Robert; Smith, Leonard A; Ahmed, S Ashraf

    2013-08-16

    Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.

  7. Anti-site defected MoS2 sheet for catalytic application

    NASA Astrophysics Data System (ADS)

    Sharma, Archana; Husain, Mushahid; Khan, Mohd. Shahid

    2018-04-01

    To prevent harmful and poisonous CO gas molecules, catalysts are needed for converting them into benign substances. Density functional theory (DFT) calculations have been used to investigate CO oxidation on the surface of MoS2 monolayer with Mo atom embedded at S-vacancy site (anti-site defect). The stronger interaction between Mo metal with O2 molecule as compared with CO molecule suggests high catalytic activity. The complete oxidation of CO is studied in a two-step procedure using Langmuir-Hinshelwood (LH) and Eley-Rideal (ER) mechanisms with a low overall energy barrier of 0.35 eV. Creation of anti-site defect makes the surface of MoS2 nanosheet catalytically active for the CO oxidation to take place.

  8. Elimination of a ligand gating site generates a supersensitive olfactory receptor.

    PubMed

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

    2016-06-21

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

  9. Elimination of a ligand gating site generates a supersensitive olfactory receptor

    PubMed Central

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

    2016-01-01

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

  10. A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions.

    PubMed

    Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Hisano, Tamao; Abe, Hideki; Tsuge, Takeharu

    2015-06-01

    Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.

  11. Effect of H2 binding on the nonadiabatic transition probability between singlet and triplet states of the [NiFe]-hydrogenase active site.

    PubMed

    Kaliakin, Danil S; Zaari, Ryan R; Varganov, Sergey A

    2015-02-12

    We investigate the effect of H2 binding on the spin-forbidden nonadiabatic transition probability between the lowest energy singlet and triplet electronic states of [NiFe]-hydrogenase active site model, using a velocity averaged Landau-Zener theory. Density functional and multireference perturbation theories were used to provide parameters for the Landau-Zener calculations. It was found that variation of the torsion angle between the terminal thiolate ligands around the Ni center induces an intersystem crossing between the lowest energy singlet and triplet electronic states in the bare active site and in the active site with bound H2. Potential energy curves between the singlet and triplet minima along the torsion angle and H2 binding energies to the two spin states were calculated. Upon H2 binding to the active site, there is a decrease in the torsion angle at the minimum energy crossing point between the singlet and triplet states. The probability of nonadiabatic transitions at temperatures between 270 and 370 K ranges from 35% to 32% for the active site with bound H2 and from 42% to 38% for the bare active site, thus indicating the importance of spin-forbidden nonadiabatic pathways for H2 binding on the [NiFe]-hydrogenase active site.

  12. Modulating the activity of protein conjugated to gold nanoparticles by site-directed orientation and surface density of bound protein.

    PubMed

    Liu, Feng; Wang, Lei; Wang, Hongwei; Yuan, Lin; Li, Jingwen; Brash, John Law; Chen, Hong

    2015-02-18

    The key property of protein-nanoparticle conjugates is the bioactivity of the protein. The ability to accurately modulate the activity of protein on the nanoparticles at the interfaces is important in many applications. In the work reported here, modulation of the activity of protein-gold nanoparticle (AuNP) conjugates by specifically orienting the protein and by varying the surface density of the protein was investigated. Different orientations were achieved by introducing cysteine (Cys) residues at specific sites for binding to gold. We chose Escherichia coli inorganic pyrophosphatase (PPase) as a model protein and used site-directed mutagenesis to generate two mutant types (MTs) with a single Cys residue on the surface: MT1 with Cys near the active center and MT2 with Cys far from the active center. The relative activities of AuNP conjugates with wild type (WT), MT1, and MT2 were found to be 44.8%, 68.8%, and 91.2% of native PPase in aqueous solution. Site-directed orientation with the binding site far from the active center thus allowed almost complete preservation of the protein activity. The relative activity of WT and MT2 conjugates did not change with the surface density of the protein, while that of MT1 increased significantly with increasing surface density. These results demonstrate that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density. Furthermore, increasing the surface density of the specifically oriented protein MT2, while having no significant effect on the specific activity of the protein, still allowed increased protein loading on the AuNP and thus increased the total protein activity. This is of great importance in the study on the interface of protein and nanoparticle and the applications for enzyme immobilization, drug delivery, and biocatalysis.

  13. Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

    PubMed

    Li, Xiao-Ping; Kahn, Peter C; Kahn, Jennifer Nielsen; Grela, Przemyslaw; Tumer, Nilgun E

    2013-10-18

    Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K(m) values and catalytic rates (k(cat)) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K(m) and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

  14. Risk Assessment for the Explosive Washout Lagoons (Site 4), Umatilla Depot Activity Hermiston, Oregon

    DTIC Science & Technology

    1992-03-01

    Activity .............................. 2 2-2 Explosive Washout Lagoons (Site 4) and Washout Plant Area ............................... 2-3 3-1 Site 4...ponds for liquid wastes from bomb-washing operations in the washout plant . The measured dimensions of the flat bottoms of the two lagoons are 30 by 80...explosives washout plant system was drained, flushed, and cleaned approximately once each week from the mid-1950s until 1965. The lagoons received all of the

  15. Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

    PubMed Central

    Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

    1994-01-01

    Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

  16. Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B.

    PubMed

    Ozcan, Ahmet; Olmez, Elif Ozkirimli; Alakent, Burak

    2013-05-01

    In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed ) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen ) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. Copyright © 2013 Wiley Periodicals, Inc.

  17. Flexibility Matters: Cooperative Active Sites in Covalent Organic Framework and Threaded Ionic Polymer.

    PubMed

    Sun, Qi; Aguila, Briana; Perman, Jason; Nguyen, Nicholas; Ma, Shengqian

    2016-12-07

    The combination of two or more reactive centers working in concert on a substrate to facilitate the reaction is now considered state of the art in catalysis, yet there still remains a tremendous challenge. Few heterogeneous systems of this sort have been exploited, as the active sites spatially separated within the rigid framework are usually difficult to cooperate. It is now shown that this roadblock can be surpassed. The underlying principle of the strategy presented here is the integration of catalytic components with excellent flexibility and porous heterogeneous catalysts, as demonstrated by the placement of linear ionic polymers in close proximity to surface Lewis acid active sites anchored on the walls of a covalent organic framework (COF). Using the cycloaddition of the epoxides and CO 2 as a model reaction, dramatic activity improvements have been achieved for the composite catalysts in relation to the individual catalytic component. Furthermore, they also clearly outperform the benchmark catalytic systems formed by the combination of the molecular organocatalysts and heterogeneous Lewis acid catalysts, while affording additional recyclability. The extraordinary flexibility and enriched concentration of the catalytically active moieties on linear polymers facilitate the concerted catalysis, thus leading to superior catalytic performance. This work therefore uncovers an entirely new strategy for designing bifunctional catalysts with double-activation behavior and opens a new avenue in the design of multicapable systems that mimic biocatalysis.

  18. NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

    PubMed Central

    Dunn, Michael F.

    2013-01-01

    Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR

  19. Examination of a Social-Networking Site Activities Scale (SNSAS) Using Rasch Analysis

    ERIC Educational Resources Information Center

    Alhaythami, Hassan; Karpinski, Aryn; Kirschner, Paul; Bolden, Edward

    2017-01-01

    This study examined the psychometric properties of a social-networking site (SNS) activities scale (SNSAS) using Rasch Analysis. Items were also examined with Rasch Principal Components Analysis (PCA) and Differential Item Functioning (DIF) across groups of university students (i.e., males and females from the United States [US] and Europe; N =…

  20. Identification and characterization of Hoxa9 binding sites in hematopoietic cells

    PubMed Central

    Huang, Yongsheng; Sitwala, Kajal; Bronstein, Joel; Sanders, Daniel; Dandekar, Monisha; Collins, Cailin; Robertson, Gordon; MacDonald, James; Cezard, Timothee; Bilenky, Misha; Thiessen, Nina; Zhao, Yongjun; Zeng, Thomas; Hirst, Martin; Hero, Alfred; Jones, Steven

    2012-01-01

    The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia. PMID:22072553