Science.gov

Sample records for active site suggests

  1. The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism

    PubMed Central

    Leiros, Hanna-Kirsti S.; Fedøy, Anita-Elin; Leiros, Ingar; Steen, Ida Helene

    2012-01-01

    Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)+-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP+, isocitrate and Mg2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251′ and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP+ into the correct orientation, which together are important for NADP+ selectivity. PMID:23650595

  2. The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism.

    PubMed

    Leiros, Hanna-Kirsti S; Fedøy, Anita-Elin; Leiros, Ingar; Steen, Ida Helene

    2012-01-01

    Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP(+), isocitrate and Mg(2+) to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251' and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP(+) into the correct orientation, which together are important for NADP(+) selectivity.

  3. A Comparison of Vanadate to a 2'-5' Linkage at the Active Site of a Small Ribozyme Suggests a Role for Water in Transition-State Stabilization

    SciTech Connect

    Torelli, A.T.; Krucinska, J.; Wedekind, J.E.

    2009-06-04

    The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 A resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2',5'-phosphodiester linkage at the site of cleavage was also solved at 2.35 A resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2',5' structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2'-O nucleophile with the scissile G+1 phosphorus. The 2',5'-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2',5' linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.

  4. Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain.

    PubMed

    Hoang, Charmaine; Hamilton, Christopher S; Mueller, Eugene G; Ferré-D'Amaré, Adrian R

    2005-08-01

    The pseudouridine synthase TruB is responsible for the universally conserved post-transcriptional modification of residue 55 of elongator tRNAs. In addition to the active site, the "thumb", a peripheral domain unique to the TruB family of enzymes, makes extensive interactions with the substrate. To coordinate RNA binding and release with catalysis, the thumb may be able to sense progress of the reaction in the active site. To establish whether there is a structural correlate of communication between the active site and the RNA-sequestering thumb, we have solved the structure of a catalytically inactive point mutant of TruB in complex with a substrate RNA, and compared it to the previously determined structure of an active TruB bound to a reaction product. Superposition of the two structures shows that they are extremely similar, except in the active site and, intriguingly, in the relative position of the thumb. Because the two structures were solved using isomorphous crystals, and because the thumb is very well ordered in both structures, the displacement of the thumb we observe likely reflects preferential propagation of active site perturbations to this RNA-binding domain. One of the interactions between the active site and the thumb involves an active site residue whose hydrogen-bonding status changes during the reaction. This may allow the peripheral RNA-binding domain to monitor progress of the pseudouridylation reaction.

  5. 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…

  6. Site Supervisors of Professional School Counseling Interns: Suggested Guidelines.

    ERIC Educational Resources Information Center

    Roberts, Walter B., Jr.; Morotti, Allan A.

    2001-01-01

    Professional school counselors operate under a myriad of working conditions, some more conducive to hosting interns than others. Potential site supervisors must have some framework from which to operate in order to provide optimum internship experiences. Guidelines are suggested that address expectations; training; mentoring; and communication…

  7. Recombination events suggest potential sites for the Huntington's disease gene.

    PubMed

    MacDonald, M E; Haines, J L; Zimmer, M; Cheng, S V; Youngman, S; Whaley, W L; Wexler, N; Bucan, M; Allitto, B A; Smith, B

    1989-08-01

    The Huntington's disease gene (HD) maps distal to the D4S10 marker in the terminal 4p16.3 subband of chromosome 4. Directed cloning has provided several DNA segments that have been grouped into three clusters on a physical map of approximately 5 X 10(6) bp in 4p16.3. We have typed RFLPs in both reference and HD pedigrees to produce a fine-structure genetic map that establishes the relative order of the clusters and further narrows the target area containing the HD gene. Despite the large number of meiotic events examined, the HD gene cannot be positioned relative to the most distal cluster. One recombination event with HD suggests that the terminal-most markers flank the disease gene; two others favor a telomeric location for the defect. Efforts to isolate the HD gene must be divided between these two distinct intervals until additional genetic data resolve the apparent contradiction in localization.

  8. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    PubMed

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease. PMID:27494228

  9. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    PubMed

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

  10. Suggested Activities for a Unit on the Middle East.

    ERIC Educational Resources Information Center

    Ediger, Marlow

    1983-01-01

    Student activities that focus on the different cultures and the history of the Middle East from Biblical times to the present are suggested. These include debates, art projects, slide shows, maps, and research problems. (IS)

  11. World War II Commemoration Committee: Fact Sheet and Suggested Activities.

    ERIC Educational Resources Information Center

    Department of Defense, Washington, DC.

    This packet suggests activities and events that school districts, schools, classes, and educational organizations can conduct to commemorate World War II. Suggestions are made to include local veterans, including those in veteran's and nursing homes and hospitals, and youth at every possible opportunity. Recognition can take the form of military…

  12. [Ute Unit with History, Suggested Activities, and Teachers' Guide.

    ERIC Educational Resources Information Center

    Montelores Studies Center, Cortez, CO.

    This curriculum unit for fourth grade students, developed by the Montelores Studies Center, Cortez, Colorado, which is funded by the Elementary and Secondary Education Act, Title III, presents a history of the Ute Indians, suggested activities for students, and a teachers' guide. The history section outlines the historical development of the Ute…

  13. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    PubMed

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  14. Sharing of Potential Nest Sites by Etheostoma olmstedi Males Suggests Mutual Tolerance in an Alloparental Species

    PubMed Central

    Stiver, Kelly A.; Wolff, Stephen H.; Alonzo, Suzanne H.

    2013-01-01

    When reproductive competitors tolerate or cooperate with one another, they may gain particular benefits, such as collectively guarding resources or attracting mates. Shared resources may be those essential to reproduction, such as a breeding site or nest. Using the tessellated darter, a species where males but not females compete over potential nest sites, we examined site use and sharing under controlled conditions of differing competitor density. Sharing was observed even when competitor density was low and individuals could have each occupied a potential nest site without same-sex sharing. Males were more likely to share a nest site with one other when the difference in size between them was larger rather than smaller. There was no evidence that female sharing was dependent on their relative size. Fish were generally more likely to use and share larger sites, in accordance with the greater relative surface area they offered. We discuss how one or both sharing males may potentially benefit, and how male sharing of potential nest sites could relate to female mating preferences. Tessellated darter males are known to provide alloparental care for eggs but this occurs without any social contact between the alloparent and the genetic father of the young. Thus, the suggestion that they may also share sites and maintain social contact with reproductive competitors highlights the importance of increased focus on the potential complexity of reproductive systems. PMID:23468853

  15. 78 FR 73518 - Notice Inviting Suggestions for New Experiments for the Experimental Sites Initiative; Federal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-06

    ... and academic success, result in shorter time to degree, and reduce student loan indebtedness. Based on... reduce reliance on student loans. Subject to the statutory restrictions and limitations of the Secretary... Notice Inviting Suggestions for New Experiments for the Experimental Sites Initiative; Federal...

  16. 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

  17. Think Texas! Suggested Activities to Help Celebrate Our Sesquicentennial.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin.

    A packet of teaching activities helps elementary and secondary teachers commemorate the sesquicentennial of Texas' independence. Activities include listening to stories about the mockingbird, bluebonnet, and pecan tree, drawing interpretations of these stories, and using a graphics tablet, light pen, or graphics software to illustrate a Texas folk…

  18. Physical activity in adolescence. A review with clinical suggestions.

    PubMed

    Lotan, Meir; Merrick, Joav; Carmeli, Eli

    2005-01-01

    Despite some inconsistencies in research methodologies, most findings support a positive correlation between participation in physical activities and well-being in adulthood. The results are consistent across the life span of both genders. Favorable connection between physical exercise to physical, psychological, emotional and educational benefits has been constantly proven. Despite such results a comparison between present to past findings show a global tendency for sedentary life style and reduced physical activities in many countries across ages and genders. There are claims that achieving an adult healthy life style is rooted in habits acquired at early ages, thus pointing at childhood and adolescence as the starting point of an active and healthy adulthood. The present article reviews the current literature and findings relating to physical activity with better health and an emphasis on adolescence. Factors correlated to participation of adolescents in physical activities are presented and some clinical issues to promote such activity are discussed. The authors strongly recommend enhanced initiation of community based easily accessed physical exercise programs, for children and adolescents.

  19. Suggestions, Resources and Activities for Teaching about Japan.

    ERIC Educational Resources Information Center

    Thomas, Paul F.

    This teacher resource packet contains a total of 28 modules for teaching about Japan at the elementary and secondary level. Activities on the Japanese family appropriate for grade 1 focus on similarities and differences, family size, family needs, and family roles. Grade 2 lessons look at the school, neighborhood, roles of children in the…

  20. Chemistry: Experiments, Demonstrations and Other Activities Suggested for Chemistry.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany. Bureau of Secondary Curriculum Development.

    This publication is a handbook used in conjunction with the course of study in chemistry developed through the New York State Education Department and The University of the State of New York. It contains experiments, demonstrations, and other activities for a chemistry course. Areas covered include the science of chemistry, the atomic structure of…

  1. Suggested Activities for Teaching Reading through the Newspaper.

    ERIC Educational Resources Information Center

    Henney, Maribeth

    The 101 activities using the newspaper for teaching reading listed in this document range from such simple tasks as having children write captions for photos from the newspaper to the more advanced and complicated assignments of learning the use of propaganda devices or the five kinds of news stories. In addition, definitions for newspaper terms…

  2. Salt site performance assessment activities

    SciTech Connect

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

    1983-01-01

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

  3. The syndrome of continuous muscle fiber activity. Evidence to suggest proximal neurogenic causation.

    PubMed

    Irani, P F; Purohit, A V; Wadia, N H

    1977-04-01

    Four patients with the syndrome of continuous muscle fiber activity were seen in a period of 6 years. Young females predominated. Remarkable improvement followed phenytoin sodium and carbamazepine administration in three patients, one of whom was "cured" within 4 years. In the remaining patient the response was inconstant. Electromyography showed abnormal spontaneous activity with diphasic and triphasic potentials appearing as doublets and multiplets. Waxing and waning was observed. D-tubocurarine and succinylcholine abolished the spontaneous activity excluding the muscle and the myoneural junction as its source. Spinal anesthesia, thiopental sodium, sleep and baclofen had no effect on it, ruling out a central source. In three patients, nerve blocks at the knee and elbow or wrist abolished this activity pointing to a proximal site of origin in the nerve somewhere between the spinal cord and the nerve block. In the remaining patient such a block significantly reduced but did not abolished the activity suggesting a dual source above and below the block. Finally successive examinations in one of our patients led us to believe that this activity may arise from different sites at varying times. It appears that regardless of the site of origin of the activity in the motor axon the counter part clinical syndrome remains the same. PMID:857572

  4. A Computational Model of the LGI1 Protein Suggests a Common Binding Site for ADAM Proteins

    PubMed Central

    Leonardi, Emanuela; Andreazza, Simonetta; Vanin, Stefano; Busolin, Giorgia; Nobile, Carlo; Tosatto, Silvio C. E.

    2011-01-01

    Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions. A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times. The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process. PMID:21479274

  5. 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

  6. 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

  7. Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

    PubMed

    Wenzl, M Verena; Beretta, Matteo; Griesberger, Martina; Russwurm, Michael; Koesling, Doris; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2011-08-01

    To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

  8. Social Networking Web Sites and Human Resource Personnel: Suggestions for Job Searches

    ERIC Educational Resources Information Center

    Roberts, Sherry J.; Roach, Terry

    2009-01-01

    Social Networking Web sites (SNWs) are now being used as reference checks by human resource personnel. For this reason, SNW users, particularly university students and other soon-to-be job applicants, should ask the following questions: Am I loading information that I want the world to see? Is this really a picture that shows me in the best light?…

  9. Spectral study of suggested Apollo sites. [proposals for financial support and the electronic spectra of pyroxenes

    NASA Technical Reports Server (NTRS)

    Mccord, T. B.

    1973-01-01

    The spectrophotometry (0.3 to 1.1 microns) of visited and proposed Apollo landing sites is presented along with proposals for financial support of the spectral study. The electronic spectra of pyroxenes is investigated along with an interpretation of telescopic spectral reflectivity curves of the moon. Reprints of published articles related to these studies are included.

  10. 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.

  11. Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade.

    PubMed

    Kinch, Lisa N; Ginalski, Krzysztof; Grishin, Nick V

    2006-01-01

    Site-2 proteases (S2Ps) form a large family of membrane-embedded metalloproteases that participate in cellular signaling pathways through sequential cleavage of membrane-tethered substrates. Using sequence similarity searches, we extend the S2P family to include remote homologs that help define a conserved structural core consisting of three predicted transmembrane helices with traditional metalloprotease functional motifs and a previously unrecognized motif (GxxxN/S/G). S2P relatives were identified in genomes from Bacteria, Archaea, and Eukaryota including protists, plants, fungi, and animals. The diverse S2P homologs divide into several groups that differ in various inserted domains and transmembrane helices. Mammalian S2P proteases belong to the major ubiquitous group and contain a PDZ domain. Sequence and structural analysis of the PDZ domain support its mediating the sequential cleavage of membrane-tethered substrates. Finally, conserved genomic neighborhoods of S2P homologs allow functional predictions for PDZ-containing transmembrane proteases in extra-cytoplasmic stress response and lipid metabolism.

  12. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

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

    1985-01-01

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

  13. Stable Isotopes Suggest Low Site Fidelity in Bar-headed Geese (Anser indicus) in Mongolia: Implications for Disease Transmission

    PubMed Central

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

    2016-01-01

    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.

  14. 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.

  15. Stable Isotopes Suggest Low Site Fidelity in Bar-headed Geese (Anser indicus) in Mongolia: Implications for Disease Transmission

    PubMed Central

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

    2016-01-01

    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. PMID:27695389

  16. Unusual Splice-Site Mutations in the RSK2 Gene and Suggestion of Genetic Heterogeneity in Coffin-Lowry Syndrome

    PubMed Central

    Zeniou, Maria; Pannetier, Solange; Fryns, Jean-Pierre; Hanauer, André

    2002-01-01

    Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation that is characterized, in male patients, by psychomotor and growth retardation and various skeletal anomalies. Typical facial changes and specific clinical and radiological hand aspects exhibited by patients are essential clues for the diagnosis. CLS is caused by mutations in a gene that is located in Xp22.2 and that encodes RSK2, a growth-factor–regulated protein kinase. RSK2 mutations are extremely heterogeneous and lead to premature termination of translation and/or loss of phosphotransferase activity. Surprisingly, among a series of 250 patients screened by single-strand conformation polymorphism (SSCP) analysis, in whom a clinical diagnosis of CLS was made, no mutations were detected in 66% (165) of the patients. To determine what proportion of these latter patients have a RSK2 mutation that has not been detected and what proportion have different disorders that are phenotypically similar to CLS, we have, in the present article, investigated, by western blot analysis and in vitro kinase assay, cell lines from 26 patients in whom no mutation was previously identified by SSCP analysis. This approach allowed us to identify seven novel RSK2 mutations: two changes in the coding sequence of RSK2, one intragenic deletion, and four unusual intronic nucleotide substitutions that do not affect the consensus GT or AG splice sites. We have also determined the nucleotide sequence of the promoter region of the RSK2 gene, and we have screened it for mutations. No disease-causing nucleotide change was identified, suggesting that mutations affecting the promoter region are unlikely to account for a large number of patients with CLS. Finally, our results provide evidence that some patients have a disease that is phenotypically very similar to CLS, which is not caused by RSK2 defects. This suggests that there are defects in either additional genes or combinations of genes that may result in a CLS

  17. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  18. 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,…

  19. 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.)

  20. 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

  1. Differential expression of active zone proteins in neuromuscular junctions suggests functional diversification.

    PubMed

    Juranek, Judyta; Mukherjee, Konark; Rickmann, Michael; Martens, Henrik; Calka, Jaroslaw; Südhof, Thomas C; Jahn, Reinhard

    2006-12-01

    Nerve terminals of the central nervous system (CNS) contain specialized release sites for synaptic vesicles, referred to as active zones. They are characterized by electron-dense structures that are tightly associated with the presynaptic plasma membrane and organize vesicle docking and priming sites. Recently, major protein constituents of active zones have been identified, including the proteins Piccolo, Bassoon, RIM, Munc13, ERCs/ELKs/CASTs and liprins. While it is becoming apparent that each of these proteins is essential for synaptic function in the CNS, it is not known to what extent these proteins are involved in synaptic function of the peripheral nervous system. Somatic neuromuscular junctions contain morphologically and functionally defined active zones with similarities to CNS synapses. In contrast, sympathetic neuromuscular varicosities lack active zone-like morphological specializations. Using immunocytochemistry at the light and electron microscopic level we have now performed a systematic investigation of all five major classes of active zone proteins in peripheral neuromuscular junctions. Our results show that somatic neuromuscular endplates contain a full complement of all active zone proteins. In contrast, varicosities of the vas deferens contain a subset of active zone proteins including Bassoon and ELKS2, with the other four components being absent. We conclude that Bassoon and ELKS2 perform independent and specialized functions in synaptic transmission of autonomic synapses.

  2. Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

    SciTech Connect

    Carlson, L.; Simon, M.; Briggs, J. A. G.; Glass, B.; Riches, J. D.; Johnson, M. C.; Muller, B.; Grunewald, K.; Krausslich, H.-G.

    2008-12-11

    Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release - akin to its role in vesicle formation - and is not restricted to severing the thin membrane tether.

  3. 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.

  4. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  5. Tractography Activation Patterns in Dorsolateral Prefrontal Cortex Suggest Better Clinical Responses in OCD DBS

    PubMed Central

    Hartmann, Christian J.; Lujan, J. Luis; Chaturvedi, Ashutosh; Goodman, Wayne K.; Okun, Michael S.; McIntyre, Cameron C.; Haq, Ihtsham U.

    2016-01-01

    Background: Medication resistant obsessive-compulsive disorder (OCD) patients can be successfully treated with Deep Brain Stimulation (DBS) which targets the anterior limb of the internal capsule (ALIC) and the nucleus accumbens (NA). Growing evidence suggests that in patients who respond to DBS, axonal fiber bundles surrounding the electrode are activated, but it is currently unknown which discrete pathways are critical for optimal benefit. Our aim was to identify axonal pathways mediating clinical effects of ALIC-NA DBS. Methods: We created computational models of ALIC-NA DBS to simulate the activation of fiber tracts and to identify connected cerebral regions. The pattern of activated axons and their cortical targets was investigated in six OCD patients who underwent ALIC-NA DBS. Results: Modulation of the right anterior middle frontal gyrus (dorsolateral prefrontal cortex) was associated with an excellent response. In contrast, non-responders showed high activation in the orbital part of the right inferior frontal gyrus (lateral orbitofrontal cortex/anterior ventrolateral prefrontal cortex). Factor analysis followed by step-wise linear regression indicated that YBOCS improvement was inversely associated with factors that were predominantly determined by gray matter activation results. Discussion: Our findings support the hypothesis that optimal therapeutic results are associated with the activation of distinct fiber pathways. This suggests that in DBS for OCD, focused stimulation of specific fiber pathways, which would allow for stimulation with lower amplitudes, may be superior to activation of a wide array of pathways, typically associated with higher stimulation amplitudes. PMID:26834544

  6. Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites.

    PubMed

    Ryves, W Jonathan; Dajani, Rana; Pearl, Laurence; Harwood, Adrian J

    2002-01-25

    Lithium inhibits (Li(+)) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg(2+)), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be(2+)) is a potent inhibitor of GSK-3 and competes for both Mg(2+) and ATP. Be(2+) also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li(+) and Be(2+) on GSK-3, we have devised a novel dual inhibition analysis. When Be(2+) and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li(+) exerts no interference with ADP inhibition or vice versa. We find, however, that Li(+) and Be(2+) do interfere with each other. These results suggest that Be(2+) competes for two distinct Mg(2+) binding sites: one is Li(+)-sensitive and the other, which is Li(+)-insensitive, binds the Mg:ATP complex.

  7. 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.

  8. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  9. The Crystal Structure of Six-transmembrane Epithelial Antigen of the Prostate 4 (Steap4), a Ferri/Cuprireductase, Suggests a Novel Interdomain Flavin-binding Site*

    PubMed Central

    Gauss, George H.; Kleven, Mark D.; Sendamarai, Anoop K.; Fleming, Mark D.; Lawrence, C. Martin

    2013-01-01

    Steap4 is a cell surface metalloreductase linked to obesity-associated insulin resistance. Initial characterization of its cell surface metalloreductase activity has been reported, but thorough biochemical characterization of this activity is lacking. Here, we report detailed kinetic analysis of the Steap4 cell surface metalloreductase activities. Steap4 shows physiologically relevant Km values for both Fe3+ and Cu2+ and retains activity at acidic pH, suggesting it may also function within intracellular organelles to reduce these metals. Flavin-dependent NADPH oxidase activity that was much greater than the equivalent Steap3 construct was observed for the isolated N-terminal oxidoreductase domain. The crystal structure of the Steap4 oxidoreductase domain was determined, providing a structural explanation for these differing activities. Structure-function work also suggested Steap4 utilizes an interdomain flavin-binding site to shuttle electrons between the oxidoreductase and transmembrane domains, and it showed that the disordered N-terminal residues do not contribute to enzymatic activity. PMID:23733181

  10. Sporicidal activity of an improved iodide formulation and suggestions regarding the biological safety cabinet.

    PubMed

    Kida, Nori

    2009-06-01

    The sporicidal activity of an improved iodide formulation based on a previously reported agent (Kida et al., 2004, tentatively designated as the KMT reagent) which is composed of 50 mM EDTA-2Na, 50 mM ferric chloride hexahydrate (FeCl3.6H2O), 50 mM potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3 with hydrochloric acid, was examined in the liquid and vapor phases. The improved iodide formulation subject to distillation (tentatively designated as the distilled KMT reagent: pH around 3) showed comparable sporicidal activity with the KMT reagent. As for the dilution effect, dilution at 1:2 showed more potent sporicidal activity than the undiluted one. It achieved complete disinfection with a treatment for 5 min at 20 degrees C and for 60 min at 5 degrees C. Even at a ratio of 1:100, the dilutions showed significant sporicidal activities at 37 degrees C. The experiment on the disinfection of the biological safety cabinet (Class II type A) as a practical possibility showed that pretreatment with 400 ml of water vapor treatment, and a mixture of 300 ml of this reagent and 150 ml of water in vapor phase achieved complete disinfection after a 24 h-decontamination process. The distilled KMT reagent may be useful for disinfecting against various contaminated materials and sites in both the liquid phase and vapor phase. PMID:19579660

  11. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  12. Computational Investigations of Trichoderma Reesei Cel7A Suggest New Routes for Enzyme Activity Improvements

    SciTech Connect

    Beckham, G. T.; Payne, C. M.; Bu, L.; Taylor, C. B.; McCabe, C.; Chu, J. W.; Himmel, M. E.; Crowley, M. F.

    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, 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.

  13. The structure of the PERK kinase domain suggests the mechanism for its activation

    SciTech Connect

    Cui, Wenjun; Li, Jingzhi; Ron, David; Sha, Bingdong

    2012-08-31

    The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK's N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the {alpha}-subunit of translation initiation factor 2 (eIF2{alpha}), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK's kinase domain has been determined to 2.8 {angstrom} resolution. The structure resembles the back-to-back dimer observed in the related eIF2{alpha} kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix {alpha}G in the C-terminal lobe, preparing the latter for eIF2{alpha} binding. The structure suggests conservation in the mode of activation of eIF2{alpha} kinases and is consistent with a 'line-up' model for PERK activation triggered by oligomerization of its luminal domain.

  14. Suggested improvements to the standard filter paper assay used to measure cellulase activity.

    PubMed

    Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar; Holtzapple, Mark

    2003-06-20

    Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized.

  15. Suggested improvements to the standard filter paper assay used to measure cellulase activity.

    PubMed

    Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar; Holtzapple, Mark

    2003-06-20

    Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized. PMID:12673775

  16. Fractal analysis reveals subclasses of neurons and suggests an explanation of their spontaneous activity.

    PubMed

    Favela, Luis H; Coey, Charles A; Griff, Edwin R; Richardson, Michael J

    2016-07-28

    The present work used fractal time series analysis (detrended fluctuation analysis; DFA) to examine the spontaneous activity of single neurons in an anesthetized animal model, specifically, the mitral cells in the rat main olfactory bulb. DFA bolstered previous research in suggesting two subclasses of mitral cells. Although there was no difference in the fractal scaling of the interspike interval series at the shorter timescales, there was a significant difference at longer timescales. Neurons in Group B exhibited fractal, power-law scaled interspike intervals, whereas neurons in Group A exhibited random variation. These results raise questions about the role of these different cells within the olfactory bulb and potential explanations of their dynamics. Specifically, self-organized criticality has been proposed as an explanation of fractal scaling in many natural systems, including neural systems. However, this theory is based on certain assumptions that do not clearly hold in the case of spontaneous neural activity, which likely reflects intrinsic cell dynamics rather than activity driven by external stimulation. Moreover, it is unclear how self-organized criticality might account for the random dynamics observed in Group A, and how these random dynamics might serve some functional role when embedded in the typical activity of the olfactory bulb. These theoretical considerations provide direction for additional experimental work. PMID:27189719

  17. Dissociation and metal-binding characteristics of yellow lichen substances suggest a relationship with site preferences of lichens

    PubMed Central

    Hauck, Markus; Jürgens, Sascha-René; Willenbruch, Karen; Huneck, Siegfried; Leuschner, Christoph

    2009-01-01

    Background and Aims Many species of lichen-forming fungi contain yellow or orange extracellular pigments belonging to the dibenzofurans (usnic acid), anthraquinones (e.g. parietin) or pulvinic acid group. These pigments are all equally efficient light screens, leading us to question the potential ecological and evolutionary significance of diversity in yellow and orange lichen substances. Here the hypothesis is tested that the different pigments differ in metal-binding characteristics, which suggest that they may contribute to adaptation to sites differing in pH and metal availability. Methods UV spectroscopy was used to study the dissociation and the pH dependence of the metal-binding behaviour of seven isolated lichen substances in methanol. Metals applied were selected macro- and micro-nutrients (Cu2+, Fe2+, Fe3+, Mg2+, Mn2+ and Zn2+). Key Results All the pigments studied are strong to moderate acids with pKa1 values between 2·8 and 4·5. Metal complexation is common in the lichen substances studied. Complexation takes place under acidic conditions with usnic acid, but under alkaline conditions with parietin and most compounds of the pulvinic acid group. The pulvinic acid derivative rhizocarpic acid forms metal complexes both in the acidic and the alkaline range. Conclusions Metal complexation by lichen substances could be a prerequisite for lichen substance-mediated control of metal uptake. Assuming such an effect at pH values where the affinity of the metal for the lichen substance is intermediate would explain the strong preference of lichens with usnic or rhizocarpic acids to acidic substrata. Moreover, it would explain the preference of lichens with parietin and some lichens with compounds of the pulvinic acid group either for nutrient-rich substrata at low pH or for calcareous substrata. PMID:18977765

  18. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1992-02-01

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

  19. 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

  20. 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

  1. Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions

    PubMed Central

    Lee, Meng-Chang; Huang, Hsin-Ju; Chang, Tzu-Hao; Huang, Hsieh-Chou; Hsieh, Shen-Yuan; Chen, Yi-Siou; Chou, Wei-Yuan; Chiang, Chiao-Hsi; Lai, Ching-Huang; Shiau, Chia-Yang

    2016-01-01

    Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency. PMID:27373565

  2. Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

    PubMed

    Lee, Meng-Chang; Huang, Hsin-Ju; Chang, Tzu-Hao; Huang, Hsieh-Chou; Hsieh, Shen-Yuan; Chen, Yi-Siou; Chou, Wei-Yuan; Chiang, Chiao-Hsi; Lai, Ching-Huang; Shiau, Chia-Yang

    2016-01-01

    Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Lead-dependent deposits in diverse synaptic vesicles: suggestive evidence for the presence of anionic binding sites

    SciTech Connect

    Sulzer, D.; Piscopo, I.; Ungar, F.; Holtzman, E.

    1987-09-01

    We have observed electron dense deposits dependent on incubation of aldehyde-fixed tissues with lead ions within synaptic vesicles of several types of neurons that differ in the neurotransmitters utilized and in the secretory granules of the adrenal medulla. Evidently, vesicle components that can interact with lead ions are widespread. A plausible explanation for the occurrence of the deposits is the presence of anionic binding sites within the vesicles. This would agree well with other biochemical, cytochemical, and immunocytochemical evidence, such as that indicating the presence of sulfated macromolecules in certain synaptic vesicles. Anionic binding sites could play significant roles by participating in processes such as Ca/sup 2 +/ storage, stabilization of pH gradients, or the control of osmotic phenomena.

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

    SciTech Connect

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

    2010-08-13

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

  7. Radiation inactivation study of aminopeptidase: probing the active site

    NASA Astrophysics Data System (ADS)

    Jamadar, V. K.; Jamdar, S. N.; Mohan, Hari; Dandekar, S. P.; Harikumar, P.

    2004-04-01

    Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2O>N 2>air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N 2O. The radicals (SCN) 2•-, Br 2•- and I 2•- inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

  8. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  9. Suggested Activities to Initiate Consumer Education in the Elementary Classroom. Centering On.

    ERIC Educational Resources Information Center

    Mainor, Nancy

    This booklet of teacher-developed and teacher-tested activities and strategies draws upon the curriculum areas of language arts, mathematics and social studies. Though prepared for fourth, fifth and sixth grade students, the material is adaptable for primary grades and can be used for group activities or as individual task cards. Activity sheets…

  10. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  11. Suggested Activities on Sociological Health Problems: Drugs, Alcoholism, Smoking for Student Teachers.

    ERIC Educational Resources Information Center

    Samalonis, Bernice

    This is a list of recommendations for a neophyte teacher for discussions with students on drugs, alcoholism, and smoking. Included are suggested readings, suggested questions for the school's drug education coordinator, recommended readings, and New York sources of information. (Related document is SP 006 468.) (JA)

  12. Electrostatic fields in the active sites of lysozymes.

    PubMed

    Sun, D P; Liao, D I; Remington, S J

    1989-07-01

    Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.

  13. 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?

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

    PubMed

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

    2016-04-22

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

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

    PubMed

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

    2016-04-22

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

  16. Comment on Birgegard and Sohlberg's (1999) suggestions for research in subliminal psychodynamic activation.

    PubMed

    Fudin, R

    2000-06-01

    Methodological changes in subliminal psychodynamic activation experiments based on the assumption that multiletter messages can be encoded automatically (Birgegard & Sohlberg, 1999) are questioned. Their contention that partial experimental messages and appropriate nonsense anagram controls (Fudin, 1986) need not be presented in every experiment is supported, with a reservation. If the difference between responses to the complete message and its control is significant in the predicted direction, then Fudin's procedure should be used. A nonsignificant difference between the response to each partial message and its control is needed to support the assumption of proponents of subliminal psychodynamic activation that successful outcomes are effected by the encoding of the meaning of a complete message. Experiments in subliminal psychodynamic activation can be improved if their methodologies take into account variables that may operate when subliminal stimuli are presented and encoded. PMID:10883752

  17. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  18. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  19. Treasury Dept. Suggests Plan to Limit Colleges' Tax Exemption for Business Activities.

    ERIC Educational Resources Information Center

    Jaschik, Scott

    1987-01-01

    Revisions of federal tax law governing the business operations of nonprofit institutions would no longer define a business activity as "related" to the organization's primary mission, and thus tax exempt, solely because it is operated for the convenience of members or students. (MSE)

  20. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

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

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

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

  2. Effect of cimetidine on catalase activity of Pseudomonas aeruginosa: a suggested mechanism of action.

    PubMed

    Masoud, Masoudeh; Ebrahimi, Farnoosh; Minai-Tehrani, Dariush

    2014-01-01

    Catalase is an important enzyme for the degradation of hydrogen peroxide in cells. Bacteria have potent catalase to deal with H2O2 in their medium culture. Any chemicals that inhibit catalase activity can be harmful for cells. Histamine H2 antagonist drugs such as cimetidine and ranitidine are used for the treatment of gastrointestinal tract disorders. The present results showed that cimetidine could inhibit the catalase activity of Pseudomonas aeruginosa in a competitive inhibition. The determination of IC50 value and Ki (6.5 μM) of cimetidine demonstrated that the enzyme binds to the drug with high affinity. Binding of the drug to the enzyme was pH-dependent and no binding was observed at basic pH (>9) and acidic pH (<6). Moreover, the imidazole ring and cyanoguanidine group of cimetidine may play an important role in inhibition by binding to Fe in heme group and glutamic acid 51 residue on the enzyme, respectively. Ranitidine had no effect on the catalase activity.

  3. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  4. An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

    SciTech Connect

    Guidinger, P.F.; Nowak, T. )

    1991-09-10

    The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 {plus minus} 0.025 M{sup {minus}1} min{sup {minus}1}. Inactivation by pyridoxal 5{prime}-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7,700 {plus minus} 860 m{sup {minus}1} min{sup {minus}1}. Treatment of the enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of k{sub obs} vs pH suggests this active-site lysine has a pK{sub a} of 8.1 and a pH-independent rate constant of inactivation of 47,700 m{sup {minus}1} min{sup {minus}1}. Proton relaxation rate measurements suggest that pyridoxal 5{prime}-phosphate modification alters binding of the phosphate-containing substrates. {sup 31}P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme {center dot}Mn{sup 2+}{center dot}substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5{prime}-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

  5. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  6. Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

    PubMed Central

    Stam, Remco; Howden, Andrew J. M.; Delgado-Cerezo, Magdalena; M. M. Amaro, Tiago M.; Motion, Graham B.; Pham, Jasmine; Huitema, Edgar

    2013-01-01

    Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility. PMID:24155749

  7. Immunocalization of telomerase in cells of lizard tail after amputation suggests cell activation for tail regeneration.

    PubMed

    Alibardi, L

    2016-02-01

    Tail amputation (autotomy) in most lizards elicits a remarkable regenerative response leading to a new although simplified tail. No information on the trigger mechanism following wounding is known but cells from the stump initiate to proliferate and form a regenerative blastema. The present study shows that telomerases are mainly activated in the nuclei of various connective and muscle satellite cells of the stump, and in other tissues, probably responding to the wound signals. Western blotting detection also indicates that telomerase positive bands increases in the regenerating blastema in comparison to the normal tail. Light and ultrastructural immunocytochemistry localization of telomerase shows that 4-14 days post-amputation in lizards immunopositive nuclei of sparse cells located among the wounded tissues are accumulating into the forming blastema. These cells mainly include fibroblasts and fat cells of the connective tissue and satellite cells of muscles. Also some immature basophilic and polychromatophilic erytroblasts, lymphoblasts and myelocytes present within the Bone Marrow of the vertebrae show telomerase localization in their nuclei, but their contribution to the formation of the regenerative blastema remains undetermined. The study proposes that one of the initial mechanisms triggering cell proliferation for the formation of the blastema in lizards involve gene activation for the production of telomerase that stimulates the following signaling pathways for cell division and migration.

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

    SciTech Connect

    Hartman, F.C.

    1991-01-01

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

  9. Antifungal activity in thrips soldiers suggests a dual role for this caste.

    PubMed

    Turnbull, Christine; Caravan, Holly; Chapman, Thomas; Nipperess, David; Dennison, Siobhan; Schwarz, Michael; Beattie, Andrew

    2012-08-23

    The social insect soldier is perhaps the most widely known caste, because it often exhibits spectacular weapons, such as highly enlarged jaws or reinforced appendages, which are used to defend the colony against enemies ranging in size from wasps to anteaters. We examined the function of the enlarged forelimbs of soldiers (both male and female) of the eusocial, gall-inhabiting insect Kladothrips intermedius, and discovered that they have little impact on their ability to repel the specialized invading thrips Koptothrips species. While the efficacy of the enlarged forelimb appears equivocal, we show that soldiers secrete strong antifungal compounds capable of controlling the specialized insect fungal pathogen, Cordyceps bassiana. Our data suggest that these thrips soldiers have evolved in response to selection by both macro- and micro-organisms. While it is unknown whether specialized fungal pathogens have been major selective agents in the evolution of the soldier caste in general, they were probably present when sociality first evolved and may have been the primordial enemies of social insects. PMID:22496077

  10. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

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

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

    SciTech Connect

    Not Available

    1990-07-01

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

  12. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

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

  13. The neural organization of semantic memory: Electrophysiological activity suggests feature-based segregation

    PubMed Central

    Sitnikova, Tatiana; West, W. Caroline; Kuperberg, Gina R.; Holcomb, Phillip J.

    2007-01-01

    Despite decades of research, it remains controversial whether semantic knowledge is anatomically segregated in the human brain. To address this question, we recorded event-related potentials (ERPs) while participants viewed pictures of animals and tools. Within the 200–600-ms epoch after stimulus presentation, animals (relative to tools) elicited an increased anterior negativity that, based on previous ERP studies, we interpret as associated with semantic processing of visual object attributes. In contrast, tools (relative to animals) evoked an enhanced posterior left-lateralized negativity that, according to prior research, might reflect accessing knowledge of characteristic motion and/or more general functional properties of objects. These results support the hypothesis of the neuroanatomical knowledge organization at the level of object features: the observed neurophysiological activity was modulated by the features that were most salient for object recognition. The high temporal resolution of ERPs allowed us to demonstrate that differences in processing animals and tools occurred specifically within the time-window encompassing semantic analysis. PMID:16129544

  14. Modelling and analysis of bacterial tracks suggest an active reorientation mechanism in Rhodobacter sphaeroides

    PubMed Central

    Rosser, Gabriel; Baker, Ruth E.; Armitage, Judith P.; Fletcher, Alexander G.

    2014-01-01

    Most free-swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. A key open question concerns varying mechanisms by which reorientation occurs. We combine mathematical modelling with analysis of a large tracking dataset to study the poorly understood reorientation mechanism in the monoflagellate species Rhodobacter sphaeroides. The flagellum on this species rotates counterclockwise to propel the bacterium, periodically ceasing rotation to enable reorientation. When rotation restarts the cell body usually points in a new direction. It has been assumed that the new direction is simply the result of Brownian rotation. We consider three variants of a self-propelled particle model of bacterial motility. The first considers rotational diffusion only, corresponding to a non-chemotactic mutant strain. Two further models incorporate stochastic reorientations, describing ‘run-and-tumble’ motility. We derive expressions for key summary statistics and simulate each model using a stochastic computational algorithm. We also discuss the effect of cell geometry on rotational diffusion. Working with a previously published tracking dataset, we compare predictions of the models with data on individual stopping events in R. sphaeroides. This provides strong evidence that this species undergoes some form of active reorientation rather than simple reorientation by Brownian rotation. PMID:24872500

  15. Effects of protein phosphatase and kinase inhibitors on the cardiac L- type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase

    PubMed Central

    1995-01-01

    We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10- 30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel. PMID:8786340

  16. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  17. 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

  18. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

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

    PubMed

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

    2015-02-24

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

  20. Using catalytic atom maps to predict the catalytic functions present in enzyme active sites.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

    Catalytic atom maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the "crowdedness" of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å rmsd of the CAM with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these CAMs were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5'-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase rmsd to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  1. Using Catalytic Atom Maps to Predict the Catalytic Functions Present in Enzyme Active Sites

    PubMed Central

    Nosrati, Geoffrey R.; Houk, K. N.

    2012-01-01

    Catalytic Atom Maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the “crowdedness” of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å RMSD of the Catalytic Atom Map with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these Catalytic Atom Maps were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5′-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase RMSD to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  2. Effects of Folding on Metalloprotein Active Sites

    NASA Astrophysics Data System (ADS)

    Winkler, Jay R.; Wittung-Stafshede, Pernilla; Leckner, Johan; Malmstrom, Bo G.; Gray, Harry B.

    1997-04-01

    Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of CuI in the unfolded protein is linear or trigonal, whereas CuII prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1,10-phenanthroline)2{}2+/+ reference system.

  3. Sixth-Grade Boys' Perceived Benefits of and Barriers to Physical Activity and Suggestions for Increasing Physical Activity

    ERIC Educational Resources Information Center

    Robbins, Lorraine B.; Talley, Henry C.; Wu, Tsu-Yin; Wilbur, JoEllen

    2010-01-01

    Interventions are needed to reduce the high overweight prevalence noted among boys in early high school. Because decreased physical activity (PA) is a factor for weight gain and a decline in boys' PA occurs across the middle school years, a need exists to intervene, as soon as boys reach middle school, to help them get adequate PA. The purpose of…

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

    SciTech Connect

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

    1986-03-20

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

  5. Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae

    PubMed Central

    Reavey, Caitlin T.; Hickman, Mark J.; Dobi, Krista C.; Botstein, David; Winston, Fred

    2015-01-01

    Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance. PMID:26281848

  6. Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae.

    PubMed

    Reavey, Caitlin T; Hickman, Mark J; Dobi, Krista C; Botstein, David; Winston, Fred

    2015-10-01

    Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.

  7. 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.

  8. Energy Crisis. Teaching Resources. A Special Publication Suggesting School Activities Which Stress Individual Responsibility Towards Energy Crisis Problems.

    ERIC Educational Resources Information Center

    Curry, Wendell; And Others

    This handbook provides public school teachers and administrators of Oregon with teaching ideas and information about the energy crisis. Suggested activities are intended to inform students (kindergarten through community college) about their responsibility toward the energy crisis and to motivate energy conservation. The handbook is divided into…

  9. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

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

    PubMed

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

    2012-09-01

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

  11. A small ribozyme with dual-site kinase activity

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Effects of Transcription Elongation Rate and Xrn2 Exonuclease Activity on RNA Polymerase II Termination Suggest Widespread Kinetic Competition.

    PubMed

    Fong, Nova; Brannan, Kristopher; Erickson, Benjamin; Kim, Hyunmin; Cortazar, Michael A; Sheridan, Ryan M; Nguyen, Tram; Karp, Shai; Bentley, David L

    2015-10-15

    The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5'PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes, suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes. PMID:26474067

  14. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

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

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)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

  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. Activation of Vago by interferon regulatory factor (IRF) suggests an interferon system-like antiviral mechanism in shrimp.

    PubMed

    Li, Chaozheng; Li, Haoyang; Chen, Yixiao; Chen, Yonggui; Wang, Sheng; Weng, Shao-Ping; Xu, Xiaopeng; He, Jianguo

    2015-01-01

    There is a debate on whether invertebrates possess an antiviral immunity similar to the interferon (IFN) system of vertebrates. The Vago gene from arthropods encodes a viral-activated secreted peptide that restricts virus infection through activating the JAK-STAT pathway and is considered to be a cytokine functionally similar to IFN. In this study, the first crustacean IFN regulatory factor (IRF)-like gene was identified in Pacific white shrimp, Litopenaeus vannamei. The L. vannamei IRF showed similar protein nature to mammalian IRFs and could be activated during virus infection. As a transcriptional regulatory factor, L. vannamei IRF could activate the IFN-stimulated response element (ISRE)-containing promoter to regulate the expression of mammalian type I IFNs and initiate an antiviral state in mammalian cells. More importantly, IRF could bind the 5'-untranslated region of L. vannamei Vago4 gene and activate its transcription, suggesting that shrimp Vago may be induced in a similar manner to that of IFNs and supporting the opinion that Vago might function as an IFN-like molecule in invertebrates. These suggested that shrimp might possess an IRF-Vago-JAK/STAT regulatory axis, which is similar to the IRF-IFN-JAK/STAT axis of vertebrates, indicating that invertebrates might possess an IFN system-like antiviral mechanism.

  18. Activation of Vago by interferon regulatory factor (IRF) suggests an interferon system-like antiviral mechanism in shrimp

    PubMed Central

    Li, Chaozheng; Li, Haoyang; Chen, Yixiao; Chen, Yonggui; Wang, Sheng; Weng, Shao-Ping; Xu, Xiaopeng; He, Jianguo

    2015-01-01

    There is a debate on whether invertebrates possess an antiviral immunity similar to the interferon (IFN) system of vertebrates. The Vago gene from arthropods encodes a viral-activated secreted peptide that restricts virus infection through activating the JAK-STAT pathway and is considered to be a cytokine functionally similar to IFN. In this study, the first crustacean IFN regulatory factor (IRF)-like gene was identified in Pacific white shrimp, Litopenaeus vannamei. The L. vannamei IRF showed similar protein nature to mammalian IRFs and could be activated during virus infection. As a transcriptional regulatory factor, L. vannamei IRF could activate the IFN-stimulated response element (ISRE)-containing promoter to regulate the expression of mammalian type I IFNs and initiate an antiviral state in mammalian cells. More importantly, IRF could bind the 5′-untranslated region of L. vannamei Vago4 gene and activate its transcription, suggesting that shrimp Vago may be induced in a similar manner to that of IFNs and supporting the opinion that Vago might function as an IFN-like molecule in invertebrates. These suggested that shrimp might possess an IRF-Vago-JAK/STAT regulatory axis, which is similar to the IRF-IFN-JAK/STAT axis of vertebrates, indicating that invertebrates might possess an IFN system-like antiviral mechanism. PMID:26459861

  19. 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.

  20. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  1. Structure-function analyses involving palindromic analogs of tritrypticin suggest autonomy of anti-endotoxin and antibacterial activities.

    PubMed

    Kaur, Kanwal J; Sarkar, Pampi; Nagpal, Sushma; Khan, Tarique; Salunke, Dinakar M

    2008-03-01

    Neutralization of invading pathogens by gene-encoded peptide antibiotics has been suggested to manifest in a variety of different modes. Some of these modes require internalization of the peptide through a pathway that involves LPS-mediated uptake of the peptide antibiotics. Many proline/tryptophan-rich cationic peptides for which this mode has been invoked do, indeed, show LPS (endotoxin) binding. If the mechanism of antibiotic action involves the LPS-mediated pathway, a positive correlation ought to manifest between the binding to LPS, its neutralization, and the bacterial killing. No such correlation was evident based on our studies involving minimal active analogs of tritrypticin. The anti-endotoxin activities of these analogs appear not to relate directly to their antibiotic potential. The two palindromic analogs of tritrypticin, NT7 (RRFPWWW) and CT7 (WWWPFRR), showed comparable antibacterial activities. However, while NT7 exhibited anti-endotoxin activity, CT7 did not. The LPS binding of two tritrypticin analogs correlated with their corresponding structures, but the antibacterial activities did not. Further structure-function analysis indicated specific structural implications of the antibacterial activity at the molecular level. Studies involving designed analogs of NT7 incorporating either rigid or flexible linkers between the specifically distanced hydrophobic and cationic clusters modulate the LPS binding. On the other hand, not knowing the target receptor for antibacterial activity is a drawback since the precise epitope for antibacterial activity is not definable. It is apparent that the anti-endotoxin and antibacterial activities represent two independent functions of tritrypticin, consistent with the emerging multifunctionality in the nature of cathelicidins.

  2. Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

    SciTech Connect

    Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard; Jardetzky, Theodore S.

    2009-05-26

    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:HLA 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.

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

    SciTech Connect

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

    1991-10-01

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

  4. 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.

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

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

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

  6. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  7. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability.

  8. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  9. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  10. Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model.

    PubMed

    Bernoux, Maud; Burdett, Hayden; Williams, Simon J; Zhang, Xiaoxiao; Chen, Chunhong; Newell, Kim; Lawrence, Gregory J; Kobe, Bostjan; Ellis, Jeffrey G; Anderson, Peter A; Dodds, Peter N

    2016-01-01

    NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling. PMID:26744216

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

    SciTech Connect

    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 positive 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.

  12. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-05-29

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  14. NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition.

    PubMed

    Mukherjee, Munia; Dutta, Kaushik; White, Mark A; Cowburn, David; Fox, Robert O

    2006-06-01

    Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered to be the primary viral determinant involved in the virus-host-cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4 viruses in the serogroup. We have determined the solution structure of LGT-E-D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT-E-D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.

  15. Multi-site Phosphorylation Regulates Bim Stability and Apoptotic Activity

    PubMed Central

    Hübner, Anette; Barrett, Tamera; Flavell, Richard A.; Davis, Roger J.

    2008-01-01

    The pro-apoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multi-site phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the anti-apoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses. PMID:18498746

  16. High adenylyl cyclase activity and in vivo cAMP fluctuations in corals suggest central physiological role

    PubMed Central

    Barott, K. L.; Helman, Y.; Haramaty, L.; Barron, M. E.; Hess, K. C.; Buck, J.; Levin, L. R.; Tresguerres, M.

    2013-01-01

    Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels. PMID:23459251

  17. 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.

  18. 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…

  19. Energy transfer at the active sites of heme proteins

    SciTech Connect

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

    1995-12-31

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

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

    PubMed

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

    2016-09-01

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

  1. Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T-cell migration

    PubMed Central

    Correa, Isabel; Plunkett, Tim; Vlad, Anda; Mungul, Arron; Candelora-Kettel, Jessica; Burchell, Joy M; Taylor–papadimitriou, Joyce; Finn, Olivera J

    2003-01-01

    MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1-specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription–polymerase chain reaction (RT–PCR) or Northern blot analysis in these cells. The profile of activated T-cell reactivity with different MUC1-specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2-based O-glycans, as opposed to the core 1 structures that dominate in the cancer-associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin-like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites. PMID:12519300

  2. Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair†

    PubMed Central

    Kanazhevskaya, Lyubov Yu.; Koval, Vladimir V.; Zharkov, Dmitry O.; Strauss, Phyllis R.; Fedorova, Olga S.

    2010-01-01

    AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis. PMID:20575528

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

    PubMed Central

    Stec, Boguslaw

    2012-01-01

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

  4. N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

    PubMed

    Hood, W F; Compton, R P; Monahan, J B

    1990-03-01

    In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

  5. Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

  6. Unbalanced activation of glutathione metabolic pathways suggests potential involvement in plant defense against the gall midge Mayetiola destructor in wheat.

    PubMed

    Liu, Xuming; Zhang, Shize; Whitworth, R Jeff; Stuart, Jeffrey J; Chen, Ming-Shun

    2015-01-01

    Glutathione, γ-glutamylcysteinylglycine, exists abundantly in nearly all organisms. Glutathione participates in various physiological processes involved in redox reactions by serving as an electron donor/acceptor. We found that the abundance of total glutathione increased up to 60% in resistant wheat plants within 72 hours following attack by the gall midge Mayetiola destructor, the Hessian fly. The increase in total glutathione abundance, however, is coupled with an unbalanced activation of glutathione metabolic pathways. The activity and transcript abundance of glutathione peroxidases, which convert reduced glutathione (GSH) to oxidized glutathione (GSSG), increased in infested resistant plants. However, the enzymatic activity and transcript abundance of glutathione reductases, which convert GSSG back to GSH, did not change. This unbalanced regulation of the glutathione oxidation/reduction cycle indicates the existence of an alternative pathway to regenerate GSH from GSSG to maintain a stable GSSG/GSH ratio. Our data suggest the possibility that GSSG is transported from cytosol to apoplast to serve as an oxidant for class III peroxidases to generate reactive oxygen species for plant defense against Hessian fly larvae. Our results provide a foundation for elucidating the molecular processes involved in glutathione-mediated plant resistance to Hessian fly and potentially other pests as well. PMID:25627558

  7. Control of active sites in selective flocculation: I -- Mathematical model

    SciTech Connect

    Behl, S.; Moudgil, B.M.; Prakash, T.S. . Dept. of Materials Science and Engineering)

    1993-12-01

    Heteroflocculation has been determined to be another major reason for loss in selectivity for flocculation process. In a mathematical model developed earlier, conditions for controlling heteroflocculation were discussed. Blocking active sites to control selective adsorption of a flocculant oil a desirable solid surface is discussed. It has been demonstrated that the lower molecular weight fraction of a flocculant which is incapable of flocculating the particles is an efficient site blocking agent. The major application of selective flocculation has been in mineral processing but many potential uses exist in biological and other colloidal systems. These include purification of ceramic powders, separating hazardous solids from chemical waste, and removal of deleterious components from paper pulp.

  8. The site of activation of factor X by cancer procoagulant.

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

    Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. A novel fMRI paradigm suggests that pedaling-related brain activation is altered after stroke

    PubMed Central

    Promjunyakul, Nutta-on; Schmit, Brian D.; Schindler-Ivens, Sheila M.

    2015-01-01

    regions were examined separately, reduced brain activation volume reached statistical significance in BA6 [p = 0.04; 4,350 (2,347) μL stroke; 6,938 (3,134) μL control] and cerebellum [p = 0.001; 4,591 (1,757) μL stroke; 8,381 (2,835) μL control]. Regardless of whether activated regions were examined together or separately, there were no significant between-group differences in brain activation intensity [p = 0.17; 1.30 (0.25)% stroke; 1.16 (0.20)% control]. Reduced volume in the stroke group was not observed during lower limb tapping and could not be fully attributed to differences in head motion or movement rate. There was a tendency for pedaling-related brain activation volume to increase with increasing work performed by the paretic limb during pedaling (p = 0.08, r = 0.525). Hence, the results of this study provide two original and important contributions. First, we demonstrated that pedaling can be used with fMRI to examine brain activation associated with lower limb movement in people with stroke. Unlike previous lower limb movements examined with fMRI, pedaling involves continuous, reciprocal, multijoint movement of both limbs. In this respect, pedaling has many characteristics of functional lower limb movements, such as walking. Thus, the importance of our contribution lies in the establishment of a novel paradigm that can be used to understand how the brain adapts to stroke to produce functional lower limb movements. Second, preliminary observations suggest that brain activation volume is reduced during pedaling post-stroke. Reduced brain activation volume may be due to anatomic, physiology, and/or behavioral differences between groups, but methodological issues cannot be excluded. Importantly, brain action volume post-stroke was both task-dependent and mutable, which suggests that it could be modified through rehabilitation. Future work will explore these possibilities. PMID:26089789

  10. A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

    PubMed

    Rashid, N; Morikawa, M; Kanaya, S; Atomi, H; Imanaka, T

    1999-02-19

    A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

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

    PubMed

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

    2015-12-29

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

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

    SciTech Connect

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

    2012-02-06

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

  13. Functional constituents of the active site of human neutrophil collagenase.

    PubMed

    Mookhtiar, K A; Wang, F; Van Wart, H E

    1986-05-01

    A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

  14. A GENOME-WIDE EXPLORATION SUGGESTS AN OLIGOGENIC MODEL OF INHERITANCE FOR THE TAFI ACTIVITY AND ITS ANTIGEN LEVELS

    PubMed Central

    Sabater-Lleal, Maria; Buil, Alfonso; Souto, Juan Carlos; Almasy, Laura; Borrell, Montserrat; Lathrop, Mark; Blangero, John; Fontcuberta, Jordi; Soria, José Manuel

    2008-01-01

    Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) is a protein that attenuates fibrinolysis potently. A considerable proportion of its variability levels is genetically determined. It has been associated with arterial and venous thrombosis. We conducted a genomewide linkage scan for genes affecting variation in plasma TAFI levels in 398 subjects from 21 extended Spanish families. The data were analyzed by a variance-component linkage method. A strong linkage was found on the long arm of Chromosome 13, near the DNA marker D13S156, where the structural gene encoding for TAFI is located. In addition, other new linkage signals were detected on chromosome regions 5p and 7q. More importantly, we performed another multipoint linkage analysis of functional TAFI conditioned on TAFI antigen levels. We detected a strong linkage signal on Chromosome 19 (LOD = 3.0, p = 0.0001) suggesting a novel QTL in this region involved in the specific functional activity of TAFI, regardless of the TAFI antigen levels. One notable aspect of this study is the identification of new QTLs that reveal a clearer picture of the genetic determinants responsible for variation in TAFI levels. Another is the replication of the linkage signal of the CPB2 gene, which confirms an important genetic determinant for TAFI antigen levels. These results strongly suggest an oligogenic mode of inheritance for TAFI, in which CPB2 gene accounts for a proportion of the variation of the phenotype together with other unknown genes that may represent potential risk factors for thrombotic disease. PMID:18563448

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

    USGS Publications Warehouse

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

    2000-01-01

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

  16. Comparison of the White-Nose Syndrome Agent Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic Enzyme Activity

    PubMed Central

    Reynolds, Hannah T.; Barton, Hazel A.

    2014-01-01

    White-nose Syndrome (WNS) is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans), is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus), which may indicate a shift in selective pressure to the detriment of P. destructans’ saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth. PMID:24466096

  17. Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity.

    PubMed

    Reynolds, Hannah T; Barton, Hazel A

    2014-01-01

    White-nose Syndrome (WNS) is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans), is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus), which may indicate a shift in selective pressure to the detriment of P. destructans' saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth. PMID:24466096

  18. Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

    PubMed

    Tolentino-Lopez, Luis; Segura-Cabrera, Aldo; Reyes-Loyola, Paola; Zimic, Mirko; Quiliano, Miguel; Briz, Veronica; Muñoz-Fernández, Angeles; Rodríguez-Pérez, Mario; Ilizaliturri-Flores, Ian; Correa-Basurto, Jose

    2013-01-01

    The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

  19. Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores of Bacillus Species Is an Atypical Aspartic Acid Protease

    PubMed Central

    Carroll, Thomas M.; Setlow, Peter

    2005-01-01

    Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease. PMID:16199582

  20. Active sites in char gasification: Final technical report

    SciTech Connect

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

    1987-09-01

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

  1. Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin

    SciTech Connect

    Dey, A.; Francis, E.J.; Adams, M.W.W.; Babini, E.; Takahashi, Y.; Fukuyama, K.; Hodgson, K.O.; Hedman, B.; Solomon, E.I.; /Stanford U., Chem. Dept. /Georgia U. /Bologna U. /Osaka U. /SLAC, SSRL

    2009-04-29

    A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.

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

    SciTech Connect

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

    2014-08-14

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

  3. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

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

    1992-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from April 1991 through September 1991. The ASEMP was established in 1989 by Solid Waste Operations (SWO) and the Environmental Sciences Division, both of Oak Ridge National Laboratory, 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. A new set of action levels was developed on the basis of a statistical analysis of background contamination. These new action levels have been used to evaluate results in this report. Results of ASEMP monitoring continue to demonstrate that no LLW (except [sup 3]H) is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II, which began in early FY 1991, was >90% complete at the end of September 1991. Results of sampling of groundwater and surface waters is presented.

  4. Maintenance of plastid RNA editing activities independently of their target sites

    PubMed Central

    Tillich, Michael; Poltnigg, Peter; Kushnir, Sergei; Schmitz-Linneweber, Christian

    2006-01-01

    RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell. PMID:16415790

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

    SciTech Connect

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

    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 connected 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.

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

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  7. Subcaste differences in neural activation suggest a prosocial role for oxytocin in eusocial naked mole-rats.

    PubMed

    Hathaway, Georgia A; Faykoo-Martinez, Mariela; Peragine, Deane E; Mooney, Skyler J; Holmes, Melissa M

    2016-03-01

    The neuropeptide oxytocin (OT) influences prosocial behavior(s), aggression, and stress responsiveness, and these diverse effects are regulated in a species- and context-specific manner. The naked mole-rat (Heterocephalus glaber) is a unique species with which to study context-dependent effects of OT, exhibiting a strict social hierarchy with behavioral specialization within the subordinate caste: soldiers are aggressive and defend colonies against unfamiliar conspecifics while workers are prosocial and contribute to in-colony behaviors such as pup care. To determine if OT is involved in subcaste-specific behaviors, we compared behavioral responses between workers and soldiers of both sexes during a modified resident/intruder paradigm, and quantified activation of OT neurons in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) using the immediate-early-gene marker c-fos co-localized with OT neurons. Resident workers and soldiers were age-matched with unfamiliar worker stimulus animals as intruders, and encounters were videorecorded and scored for aggressive behaviors. Colony-matched controls were left in their home colony for the duration of the encounters. Brains were extracted and cell counts were conducted for OT immunoreactive (ir), c-fos-ir, and percentage of OT-c-fos double-labeled cells. Results indicate that resident workers were less aggressive but showed greater OT neural activity than soldiers. Furthermore, a linear model including social treatment, cortisol, and subcaste revealed that subcaste was the only significant predictor of OT-c-fos double-labeled cells in the PVN. These data suggest that in naked mole-rats OT promotes prosocial behaviors rather than aggression and that even within subordinates status exerts robust effects on brain and behavior.

  8. Site specific rationale for technical impracticability of active groundwater restoration at a former manufactured gas plant site

    SciTech Connect

    Logan, C.M.; Walden, R.H.; MacFarlane, I.D.

    1995-12-31

    The National Contingency Plan (40 CFR Part 300 ) requires that remedial strategies must, at minimum, protect human health and the environment and meet applicable and relevant or appropriate requirements (ARARs). Where groundwater is impacted, maximum contaminant levels (MCLs) and maximum contaminant level goals (MCLGs) set under the Safe Drinking Water Act are often used as ARARs, whether or not the aquifer is a reasonably anticipated future source of drinking water. The US Environmental Protection Agency now recognizes the difficulty of groundwater restoration at sites where dense nonaqueous phase liquids are present, particularly in certain complex hydrogeological settings (EPA 1993). However, demonstration of impracticability generally does not occur until active remediation (e.g., pump and treat) has been shown to be ineffective. A case study of a former manufactured gas plant (MGP) is used to demonstrate how physical and chemical properties of the aquifer and coal tar, the major waste product from MGP sites, influence the feasibility of active restoration. Field characterization investigations, laboratory studies, and groundwater modeling are integrated into a demonstration following EPA guidelines. Laboratory studies included microbiological characterization and natural biodegradation and suggest that intrinsic bioremediation is occurring at this site. This work will be useful as EPA continues to develop presumptive remedies for cleanup under Superfund.

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

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    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–carbon 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 reducing

  10. Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

    PubMed Central

    Mann, K P; Weiss, E A; Nevins, J R

    1993-01-01

    The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection. Images PMID:8384308

  11. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

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

    SciTech Connect

    1981-02-27

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

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

    SciTech Connect

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

    2008-11-10

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

  14. Crystal Structures of the Response Regulator DosR From Mycobacterium Tuberculosis Suggest a Helix Rearrangement Mechanism for Phosphorylation Activation

    SciTech Connect

    Wisedchaisri, G.; Wu, M.; Sherman, D.R.; Hol, W.G.J.

    2009-05-26

    The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 {angstrom} resolution and its C-terminal DNA-binding domain at 1.7 {angstrom} resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected ({beta}{alpha}){sub 4} topology instead of the canonical ({beta}{alpha}){sub 5} fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix {alpha}10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix {alpha}10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.

  15. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

  16. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  17. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  18. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

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

    2013-04-24

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

  19. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  20. The active site of low-temperature methane hydroxylation in iron-containing zeolites.

    PubMed

    Snyder, Benjamin E R; Vanelderen, Pieter; Bols, Max L; Hallaert, Simon D; Böttger, Lars H; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2016-08-18

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(ii), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species-α-Fe(ii) and α-O-are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive 'spectator' iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(ii) to be a mononuclear, high-spin, square planar Fe(ii) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(iv)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function-producing what is known in the context of metalloenzymes as an 'entatic' state-might be a useful way to tune the activity of heterogeneous catalysts. PMID:27535535

  1. The active site of low-temperature methane hydroxylation in iron-containing zeolites

    NASA Astrophysics Data System (ADS)

    Snyder, Benjamin E. R.; Vanelderen, Pieter; Bols, Max L.; Hallaert, Simon D.; Böttger, Lars H.; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A.; Sels, Bert F.; Solomon, Edward I.

    2016-08-01

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(II), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species—α-Fe(II) and α-O—are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive ‘spectator’ iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(II) to be a mononuclear, high-spin, square planar Fe(II) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(IV)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function—producing what is known in the context of metalloenzymes as an ‘entatic’ state—might be a useful way to tune the activity of heterogeneous catalysts.

  2. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

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

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

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

    PubMed

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

    2015-07-21

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

  4. 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.

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

    PubMed Central

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

    2008-01-01

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

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

    SciTech Connect

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

    1986-03-01

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

  7. 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

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

    SciTech Connect

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

    2008-04-02

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

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

    SciTech Connect

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

    1992-02-01

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

  10. Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France).

    PubMed

    Lacroix, Camille; Richard, Gaëlle; Seguineau, Catherine; Guyomarch, Julien; Moraga, Dario; Auffret, Michel

    2015-05-01

    Brest harbor (Bay of Brest, Brittany, France) has a severe past of anthropogenic chemical contamination, but inputs tended to decrease, indicating a reassessment of its ecotoxicological status should be carried out. Here, native and caged mussels (Mytilus spp.) were used in combination to evaluate biological effects of chronic chemical contamination in Brest harbor. Polycyclic aromatic hydrocarbon (PAH) contamination was measured in mussel tissues as a proxy of harbor and urban pollution. Biochemical biomarkers of xenobiotic biotransformation, antioxidant defenses, generation of reducing equivalents, energy metabolism and oxidative damage were studied in both gills and digestive glands of native and caged mussels. In particular, activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), NADP-dependent isocitrate dehydrogenase (IDP), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) were measured and lipid peroxidation was assessed by malondialdehyde (MDA) quantification. In addition, a condition index was calculated to assess the overall health of the mussels. Moderate PAH contamination was detected in digestive glands of both native and caged individuals from the exposed site. Modulations of biomarkers were detected in digestive glands of native harbor mussels indicating the presence of a chemical pressure. In particular, results suggested increased biotransformation (GST), antioxidant defenses (CAT), NADPH generation (IDP) and gluconeogenesis (PEPCK), which could represent a coordinated response against chemically-induced cellular stress. Lipid peroxidation assessment and condition index indicated an absence of acute stress in the same mussels suggesting metabolic changes could, at least partially, offset the negative effects of contamination. In caged mussels, only GR was found modulated compared to non-exposed mussels but significant differences in

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

    PubMed Central

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

    2015-01-01

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

  12. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  13. Identification of active sites in gold-catalyzed hydrogenation of acrolein.

    PubMed

    Mohr, Christian; Hofmeister, Herbert; Radnik, Jörg; Claus, Peter

    2003-02-19

    The active sites of supported gold catalysts, favoring the adsorption of C=O groups of acrolein and subsequent reaction to allyl alcohol, have been identified as edges of gold nanoparticles. After our recent finding that this reaction preferentially occurs on single crystalline particles rather than multiply twinned ones, this paper reports on a new approach to distinguish different features of the gold particle morphology. Elucidation of the active site issue cannot be simply done by varying the size of gold particles, since the effects of faceting and multiply twinned particles may interfere. Therefore, modification of the gold particle surface by indium has been used to vary the active site characteristics of a suitable catalyst, and a selective decoration of gold particle faces has been observed, leaving edges free. This is in contradiction to theoretical predictions, suggesting a preferred occupation of the low-coordinated edges of the gold particles. On the bimetallic catalyst, the desired allyl alcohol is the main product (selectivity 63%; temperature 593 K, total pressure p(total) = 2 MPa). From the experimentally proven correlation between surface structure and catalytic behavior, the edges of single crystalline gold particles have been identified as active sites for the preferred C=O hydrogenation. PMID:12580618

  14. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  18. The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

    PubMed

    Li, Jun; Machius, Mischa; Chuang, Jacinta L; Wynn, R Max; Chuang, David T

    2007-04-20

    A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction. PMID:17329260

  19. Nicotinic Activity of Arecoline, the Psychoactive Element of "Betel Nuts", Suggests a Basis for Habitual Use and Anti-Inflammatory Activity.

    PubMed

    Papke, Roger L; Horenstein, Nicole A; Stokes, Clare

    2015-01-01

    Habitual chewing of "betel nut" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6-10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline's activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts.

  20. Nicotinic Activity of Arecoline, the Psychoactive Element of "Betel Nuts", Suggests a Basis for Habitual Use and Anti-Inflammatory Activity

    PubMed Central

    Papke, Roger L.; Horenstein, Nicole A.; Stokes, Clare

    2015-01-01

    Habitual chewing of "betel nut" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6–10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline's activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts. PMID:26488401

  1. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  2. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    PubMed Central

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  3. 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

  4. Barriers to and Suggestions for a Healthful, Active Lifestyle as Perceived by Rural and Urban Costa Rican Adolescents

    ERIC Educational Resources Information Center

    Monge-Rojas, Rafael; Garita-Arce, Carlos; Sanchez-Lopez, Marta; Colon-Ramos, Uriyoan

    2009-01-01

    Objective: To assess the perceptions of rural and urban Costa Rican adolescents regarding which barriers and motivators affect their adoption of an active lifestyle. Design: Data were collected in focus group discussions. Participants: 108 male and female adolescents aged 12 to 18 from the 7th to 11th grades. Setting: Two urban and 1 rural high…

  5. Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-(p-(fluorosulfonyl)benzoyl)adenosine

    SciTech Connect

    Phelps, D.C.; Hatefi, Y.

    1985-07-02

    Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-(p-(fluorosulfonyl)benzoyl)adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. (/sup 3/H)FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of (3H)FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by (/sup 14/C)DCCD. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

  6. Evidence for Oxygen Binding at the Active Site of Particulate Methane Monooxygenase

    PubMed Central

    Culpepper, Megen A.; Cutsail, George E.; Hoffman, Brian M.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α3β3γ3 trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit, denoted spmoB, indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O2 and H2O2 or pMMO with H2O2, an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to that of the μ-η2:η2-peroxo CuII 2 species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O2 binding to the proposed active site. Reaction of the 345 nm species with CH4 results in disappearance of the spectroscopic feature, suggesting that this O2 intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site. PMID:22540911

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

    PubMed

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

    2015-05-01

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

  8. Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

    PubMed

    Izumi, T; Malecki, J; Chaudhry, M A; Weinfeld, M; Hill, J H; Lee, J C; Mitra, S

    1999-03-19

    The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure. PMID:10074406

  9. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  10. Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase.

    PubMed Central

    Robey, E A; Schachman, H K

    1985-01-01

    Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains. However, the precise locations of the active sites and of the residues involved in catalysis are not known. To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue. One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis. Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer. The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers. The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers. The results constitute strong evidence for shared active sites in aspartate transcarbamoylase. Images PMID:3881763

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

    SciTech Connect

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

    2014-10-02

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

  12. A Drosophila model of GSS syndrome suggests defects in active zones are responsible for pathogenesis of GSS syndrome.

    PubMed

    Choi, Jin-Kyu; Jeon, Yong-Chul; Lee, Dae-Weon; Oh, Jae-Min; Lee, Hyun-Pil; Jeong, Byung-Hoon; Carp, Richard I; Koh, Young Ho; Kim, Yong-Sun

    2010-11-15

    We have established a Drosophila model of Gerstmann-Sträussler-Scheinker (GSS) syndrome by expressing mouse prion protein (PrP) having leucine substitution at residue 101 (MoPrP(P101L)). Flies expressing MoPrP(P101L), but not wild-type MoPrP (MoPrP(3F4)), showed severe defects in climbing ability and early death. Expressed MoPrP(P101L) in Drosophila was differentially glycosylated, localized at the synaptic terminals and mainly present as deposits in adult brains. We found that behavioral defects and early death of MoPrP(P101L) flies were not due to Caspase 3-dependent programmed cell death signaling. In addition, we found that Type 1 glutamatergic synaptic boutons in larval neuromuscular junctions of MoPrP(P101L) flies showed significantly increased numbers of satellite synaptic boutons. Furthermore, the amount of Bruchpilot and Discs large in MoPrP(P101L) flies was significantly reduced. Brains from scrapie-infected mice showed significantly decreased ELKS, an active zone matrix marker compared with those of age-matched control mice. Thus, altered active zone structures at the molecular level may be involved in the pathogenesis of GSS syndrome in Drosophila and scrapie-infected mice.

  13. An interactive activation and competition model of person knowledge, suggested by proactive interference by traits spontaneously inferred from behaviours.

    PubMed

    Wang, Yuanbo E; Higgins, Nancy C; Uleman, James S; Michaux, Aaron; Vipond, Douglas

    2016-03-01

    People unconsciously and unintentionally make inferences about others' personality traits based on their behaviours. In this study, a classic memory phenomenon--proactive interference (PI)--is for the first time used to detect spontaneous trait inferences. PI should occur when lists of behaviour descriptions, all implying the same trait, are to be remembered. Switching to a new trait should produce 'release' from proactive interference (or RPI). Results from two experiments supported these predictions. PI and RPI effects are consistent with an interactive activation and competition model of person perception (e.g., McNeill & Burton, 2002, J. Exp. Psychol., 55A, 1141), which predicts categorical organization of social behaviours based on personality traits. Advantages of this model are discussed. PMID:26096621

  14. Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm.

    PubMed

    Díaz-Pérez, E; Thomas, P; Meizel, S

    1988-11-01

    It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Anthropogenic and temporal components in a complex trigger of type 1 diabetes suggest the active participation of antipyretics.

    PubMed

    Veteikis, Darijus

    2016-08-01

    Tremendous efforts in research without a conclusion on the cause of type 1 diabetes allow the presumption that there is still a blind spot in the development of T1D that is not covered by current hypotheses. The review of geographical knowledge suggests that there is a well-expressed anthropogenic element within the complex environmental trigger of T1D. On the other hand, the initiation of T1D's directed autoimmunity is temporally related to the organism's immune response, induced by entero-viruses, most expectedly. Consequently, the searched for anthropogenic environmental factor is a player temporally linked to enteroviral infections. This paper discusses the participation of antipyretic medicines, and especially paracetamol, with a whole century's history of growing sales and popularity, including indirect influence through phenacetin during the first half of the 20th century. As proposed by several independent studies, the use of pharmaceuticals to reduce fever may counteract with the protective features of the immune system and create favourable conditions for a virus to spread within the organism and damage specific tissue. A preliminary comparison of paracetamol sales with the incidence of T1D data in Lithuania and the other countries in the North-eastern Baltic region supports this hypothesis. PMID:27372871

  16. 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.

  17. Predicted hexameric structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site during type IV secretion

    PubMed Central

    Middleton, Rebecca; Sjölander, Kimmen; Krishnamurthy, Nandini; Foley, Jonathan; Zambryski, Patricia

    2005-01-01

    The Agrobacterium T-DNA transporter belongs to a growing class of evolutionarily conserved transporters, called type IV secretion systems (T4SSs). VirB4, 789 aa, is the largest T4SS component, providing a rich source of possible structural domains. Here, we use a variety of bioinformatics methods to predict that the C-terminal domain of VirB4 (including the Walker A and B nucleotide-binding motifs) is related by divergent evolution to the cytoplasmic domain of TrwB, the coupling protein required for conjugative transfer of plasmid R388 from Escherichia coli. This prediction is supported by detailed sequence and structure analyses showing conservation of functionally and structurally important residues between VirB4 and TrwB. The availability of a solved crystal structure for TrwB enables the construction of a comparative model for VirB4 and the prediction that, like TrwB, VirB4 forms a hexamer. These results lead to a model in which VirB4 acts as a docking site at the entrance of the T4SS channel and acts in concert with VirD4 and VirB11 to transport substrates (T-strand linked to VirD2 or proteins such as VirE2, VirE3, or VirF) through the T4SS. PMID:15668378

  18. 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. PMID:26838013

  19. 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.

  20. Characterization of the active site of chloroperoxidase using physical techniques

    SciTech Connect

    Hall, K.S.

    1986-01-01

    Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.

  1. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

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

    PubMed

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

    2016-01-01

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

  3. 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.

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

    PubMed

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

    2014-06-01

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

  5. Molecular multiproxy analysis of ancient root systems suggests strong alteration of deep subsoil organic matter by rhizomicrobial activity

    NASA Astrophysics Data System (ADS)

    Gocke, Martina; Huguet, Arnaud; Derenne, Sylvie; Kolb, Steffen; Wiesenberg, Guido L. B.

    2013-04-01

    decreasing contents of archeal GDGTs from rhizolith via rhizosphere towards root-free loess. Furthermore, the bacterial fingerprint revealed - similar to modern root systems - higher taxonomic diversity in rhizosphere compared to rhizoliths and reference loess. This argues for microorganisms benefiting from root deposits and exudates. Highest concentrations of branched GDGTs in rhizoliths suggest that their source organisms feed on root remains. Incorporation of rhizomicrobial remains as represented by RNA and GDGTs usually affected the sediment at maximum to a distance of 2-3 cm from the former root. FA contents in rhizosphere showed strong scatter and were in part depleted compared to reference loess or, especially in deeper transects, enriched. This indicates the presence of degradation products originating from former rhizosphere processes. Especially at larger depth not affected by modern pedogenic processes, portions of mainly microbial derived C16 homologues were higher in rhizosphere loess up to distances of 10 cm, revealing that the possible extension of the rhizosphere was underestimated so far. In Corg poor subsoil, the occurence of diverse rhizosphere microorganisms and degradation processes even in several centimeters distant from roots point to a strong alteration of OM, possibly contributing to carbon mineralisation.

  6. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  7. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    D Gallagher; S Kim; H Robinson; P Reddy

    2011-12-31

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV) - two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  8. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    Gallagher, D.T.; Robinson, H.; Kim, S.-K.; Reddy, P. T.

    2011-01-21

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)-two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  9. Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.

    PubMed

    Pieri, Myrtani; Hall, Dashiell; Price, Richard; Bailey, Patrick; Meredith, David

    2008-01-01

    The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pH(out) from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pH(out) 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.

  10. A Relaxed Active Site After Exon Ligation by the Group I Intron

    SciTech Connect

    Lipchock,S.; Strobel, S.

    2008-01-01

    During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

  11. Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

    PubMed Central

    Herschlag, Daniel

    2016-01-01

    Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes. PMID:27501145

  12. Free energy simulations of active-site mutants of dihydrofolate reductase.

    PubMed

    Doron, Dvir; Stojković, Vanja; Gakhar, Lokesh; Vardi-Kilshtain, Alexandra; Kohen, Amnon; Major, Dan Thomas

    2015-01-22

    This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops. PMID:25382260

  13. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  14. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

  15. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

  16. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

  17. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

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

    SciTech Connect

    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.

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

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

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

  20. Control of active sites in selective flocculation: III -- Mechanism of site blocking

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    It has been shown in Parts I and II of this paper that heteroflocculation can be controlled by poisoning the sites for flocculant adsorption using a site blocking agent (SBA). An efficient SBA was determined to be the lower molecular weight fraction of the flocculant. In this paper, the underlying mechanism of SBA action is described. Also, the mathematical model detailed in Part I is used to determine the effect of different SBAs on apatite-dolomite separation efficiency. It has been demonstrated that the depression in flocculation is directly related to the site blocking parameter ([bar [Phi

  1. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

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

  2. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible.

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

    SciTech Connect

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

    2010-01-08

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

  4. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible. PMID:27344491

  5. Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor.

    PubMed

    Bos, Ineke G A; Lubbers, Yvonne T P; Eldering, Eric; Abrahams, Jan Pieter; Hack, C Erik

    2004-06-01

    The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.

  6. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  7. Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

    Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

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

  9. Control of active sites in selective flocculation: II -- Role of site blocking agents

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Control of heteroflocculation using a lower molecular weight fraction of the flocculant as a site blocking agent is demonstrated in the apatite-dolomite-polyethylene oxide system. The most effective SBA (site blocking agent) was determined to be the highest molecular weight fraction of the flocculant itself which was not capable of flocculating any of the components of the mixture. In the presence of the SBA, flocculant adsorption decreased significantly on apatite particles, thereby inhibiting coflocculation.

  10. The C-terminal tail inhibitory phosphorylation sites of PTEN regulate its intrinsic catalytic activity and the kinetics of its binding to phosphatidylinositol-4,5-bisphosphate.

    PubMed

    Chia, Yeong-Chit Joel; Catimel, Bruno; Lio, Daisy Sio Seng; Ang, Ching-Seng; Peng, Benjamin; Wu, Hong; Zhu, Hong-Jian; Cheng, Heung-Chin

    2015-12-01

    Dephosphorylation of four major C-terminal tail sites and occupancy of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]-binding site of PTEN cooperate to activate its phospholipid phosphatase activity and facilitate its recruitment to plasma membrane. Our investigation of the mechanism by which phosphorylation of these C-terminal sites controls the PI(4,5)P2-binding affinity and catalytic activity of PTEN resulted in the following findings. First, dephosphorylation of all four sites leads to full activation; and phosphorylation of any one site significantly reduces the intrinsic catalytic activity of PTEN. These findings suggest that coordinated inhibition of the upstream protein kinases and activation of the protein phosphatases targeting the four sites are needed to fully activate PTEN phosphatase activity. Second, PI(4,5)P2 cannot activate the phosphopeptide phosphatase activity of PTEN, suggesting that PI(4,5)P2 can only activate the phospholipid phosphatase activity but not the phosphoprotein phosphatase activity of PTEN. Third, dephosphorylation of all four sites significantly decreases the affinity of PTEN for PI(4,5)P2. Since PI(4,5)P2 is a major phospholipid co-localizing with the phospholipid- and phosphoprotein-substrates in plasma membrane, we hypothesise that the reduced affinity facilitates PTEN to "hop" on the plasma membrane to dephosphorylate these substrates. PMID:26471078

  11. 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

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

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    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 on 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, in

  13. Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase.

    PubMed

    Vedula, L Sangeetha; Cane, David E; Christianson, David W

    2005-09-27

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group. PMID:16171386

  14. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase

    SciTech Connect

    Vedula,L.; Cane, D.; Christianson, D.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PPi bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  15. Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II.

    PubMed

    Deupi, Xavier; Edwards, Patricia; Singhal, Ankita; Nickle, Benjamin; Oprian, Daniel; Schertler, Gebhard; Standfuss, Jörg

    2012-01-01

    G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y(6.40) constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223(5.58) in helix 5, Y306(7.53) of the NPxxY motif and R135(3.50) of the E(D)RY motif, highly conserved residues of the G protein binding site.

  16. Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

    PubMed

    Blomberg, Margareta R A

    2016-01-26

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  17. Phase I and II liver enzyme activities in juvenile alligators (Alligator mississippiensis) collected from three sites in the Kissimmee-Everglades drainage, Florida (USA).

    PubMed

    Gunderson, M P; Oberdörster, E; Guillette, L J

    2004-10-01

    We examined CYP1A (measured using hepatic EROD and MROD activities) and glutathione-S-transferase (GST) activities in juvenile alligators (Alligator mississippiensis) collected from three sites with varying contamination in the Kissimmee-Everglades drainage in south Florida. We hypothesized that contaminants present in areas with intermediate or higher contaminant concentrations would alter hepatic enzyme activities in juvenile alligators from those sites when compared to hepatic enzyme activity in animals from the area with the least contamination. EROD activity was found to be higher in animals from the site with lower reported levels of contamination relative to those from the site with the highest reported contamination suggesting an inhibition of CYP1A expression or activity. No differences among animals from the three sites were observed for hepatic MROD and GST activities. A significant negative relationship between EROD, MROD, and GST activities and body size was exhibited in alligators from the site with the lowest contamination. No relationship between body size and hepatic enzyme activity was found in animals from the sites with intermediate and higher contamination, suggesting that contaminants present at these sites act to alter this relationship. No correlation was observed in this study between plasma steroid concentrations (estradiol-17 beta or testosterone) and hepatic EROD, MROD, or GST activities.

  18. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    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.

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

    PubMed Central

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

    2014-01-01

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

  20. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  1. A Dynamic Zn Site in Helicobacter pylori HypA: A Potential Mechanism for Metal-Specific Protein Activity

    SciTech Connect

    Kennedy,D.; Herbst, R.; Iwig, J.; Chivers, P.; Maroney, M.

    2007-01-01

    HypA is an accessory protein and putative metallochaperone that is critical for supplying nickel to the active site of NiFe hydrogenases. In addition to binding Ni(II), HypA is known to contain a Zn site that has been suggested to play a structural role. X-ray absorption spectroscopy has been used to show that the Zn site changes structure upon binding nickel, from a S{sub 3}(O/N)-donor ligand environment to an S{sub 4}-donor ligand environment. This provides a potential mechanism for discriminating Ni(II) from other divalent metal ions. The Ni(II) site is shown to be a six-coordinate complex composed of O/N-donors including two histidines. As such, it resembles the nickel site in UreE, a nickel metallochaperone involved in nickel incorporation into urease.

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

    SciTech Connect

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    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 of 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.

  3. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    SciTech Connect

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-10-10

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

  4. An ionizable active-site tryptophan imparts catalase activity to a peroxidase core.

    PubMed

    Loewen, Peter C; Carpena, Xavi; Vidossich, Pietro; Fita, Ignacio; Rovira, Carme

    2014-05-21

    Catalase peroxidases (KatG's) are bifunctional heme proteins that can disproportionate hydrogen peroxide (catalatic reaction) despite their structural dissimilarity with monofunctional catalases. Using X-ray crystallography and QM/MM calculations, we demonstrate that the catalatic reaction of KatG's involves deprotonation of the active-site Trp, which plays a role similar to that of the distal His in monofunctional catalases. The interaction of a nearby mobile arginine with the distal Met-Tyr-Trp essential adduct (in/out) acts as an electronic switch, triggering deprotonation of the adduct Trp.

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

    SciTech Connect

    Daley, Thomas; Niu, Fenglin; Silver, Paul G.; Daley, Thomas M.; Cheng, Xin; Majer, Ernest L.

    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 predicted 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.

  6. Alternative 3' splice acceptor sites modulate enzymic activity in derivative alleles of the maize bronze1-mutable 13 allele.

    PubMed Central

    Okagaki, R J; Sullivan, T D; Schiefelbein, J W; Nelson, O E

    1992-01-01

    The defective Suppressor-mutator (dSpm)-induced allele bronze1-mutable 13 (bz1-m13) and many of its derivative alleles are leaky mutants with measurable levels of flavonol O3-glucosyltransferase activity. This activity results from splicing at acceptor site-1, one of two cryptic 3' splice sites within the dSpm insertion in bz1-m13. In this study, splicing in bz1-m13 change-in-state (CS) alleles CS-3 and CS-64 was shown to be altered from bz1-m13; previous work found altered splicing in CS-9. CS-64 is a null allele and lacks the acceptor site-1-spliced transcript because this site is deleted. CS-3 and CS-9 had increased levels of the acceptor site-1 transcript relative to bz1-m13 and increased enzymic activities. A deletion in CS-9 altered splicing by eliminating acceptor site-2. Both acceptor sites were intact in CS-3, but a deletion removed most of a 275-bp GC-rich sequence in dSpm. This suggests that GC-rich sequences affect splicing and is consistent with models postulating a role for AU content in the splicing of plant introns. Splicing does not necessarily occur, however, at the junction of AU-rich intron sequences and GC-rich exon sequences. PMID:1477558

  7. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

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

  8. The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

    PubMed

    Fukumoto, Mitsuki; Kudou, Daizou; Murano, Shouko; Shiba, Tomoo; Sato, Dan; Tamura, Takashi; Harada, Shigeharu; Inagaki, Kenji

    2012-01-01

    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

  9. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    SciTech Connect

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G. . E-mail: wolfd@md.huji.ac.il

    2006-10-10

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant ({delta}UL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the {delta}UL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or {delta}UL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

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

    SciTech Connect

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

    2010-07-06

    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 primarily 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.

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

    SciTech Connect

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

    1994-09-01

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

  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. Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

    SciTech Connect

    Vlahos, C.J.

    1987-01-01

    The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with (/sup 14/C)pyruvate in the presence of NaCNBH/sub 3/ results in the incorporation of one mol of /sup 14/C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with (3-/sup 14/C)bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

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

  15. Parameterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2012-12-01

    Thermokarst features are thought to be an important mechanism for landscape change in permafrost-dominated cold regions, but few such features have been incorporated into full featured landscape models. The root of this shortcoming is that historic observations are not detailed enough to parameterize a model, and the models typically do not include the relevant processes for thermal erosion. A new, dynamic thermokarst feature has been identified at the Caribou-Poker Creek Research Watershed (CPCRW) in the boreal forest of Interior Alaska. Located adjacent to a traditional use trail, this feature terminates directly in Caribou Creek. Erosion within the feature is driven predominantly by fluvial interflow. CPCRW is a Long-Term Ecological Research site underlain by varying degrees of relatively warm, discontinuous permafrost. This poster will describe the suite of measurements that have been undertaken to parameterize the ERODE model for this site, including thorough surveys, time lapse- and aerial photography, and 3-D structure from motion algorithms.

  16. 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…

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

    Not Available

    1993-01-27

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

  19. Associations between Restriction Site Polymorphism and Enzyme Activity Variation for Esterase 6 in Drosophila Melanogaster

    PubMed Central

    Game, A. Y.; Oakeshott, J. G.

    1990-01-01

    Thirty-five nucleotide polymorphisms were found in a 21.5-kbp region including the Est6 locus among 42 isoallelic lines extracted from a single natural population of Drosophila melanogaster. The heterozygosity per nucleotide pair was estimated to be 0.010 overall, but was lower in sequences hybridizing to transcripts than in those not hybridizing to transcripts. Eleven of 36 pairwise comparisons among the nine most common polymorphisms showed significant gametic disequilibrium. Four of these polymorphisms were also significantly associated with the major EST6-F/EST6-S allozyme polymorphism. Significant disequilibrium was generally restricted to polymorphisms less than 1-2 kbp apart. Previously reported measures of EST6 activity in virgin adult females proved not to be significantly associated with any of the six most common nucleotide polymorphisms located in the Est6 coding region or the 1.5 kbp immediately 5'. However, the 11 haplotypes for five of these polymorphisms that lie in the 1.5-kbp 5' region could explain about half of the previously reported variation among the lines for both EST6 activity and the amount of EST6 protein in virgin adult males. One particular polymorphism, for a RsaI site 530 bp 5' of the initiation codon, could explain 21% of the male activity variation among lines. This site is embedded in a large palindrome and we suggest that sequences including or close to this site may be involved in the regulation of EST6 synthesis in the ejaculatory duct of the adult male. PMID:1981760

  20. Identification of ice nucleation active sites on feldspar dust particles.

    PubMed

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

    2015-03-19

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

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2015-07-13

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

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

    PubMed

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

    2015-07-13

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

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

    PubMed

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

    2016-07-01

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

  5. Sediment biogeochemistry and microbial activity at natural hydrocarbon seeps and at sites impacted by anthropogenic hydrocarbon discharges

    NASA Astrophysics Data System (ADS)

    Joye, S. B.; Sibert, R.; Battles, J.; Fields, L.; Kleindienst, S.; Crespo-Medina, M.; Hunter, K.; Meile, C. D.; Montoya, J. P.

    2013-12-01

    Natural hydrocarbon seeps occur along the seafloor where geologic faults facilitate transfer of deeply sourced fluids enriched in gas, oil, and dissolved organic matter through shallow sediments and into the water column. At natural seeps, microbial populations specialize in hydrocarbon degradation and rates of microbial activity, including sulfate reduction and anaerobic oxidation of methane, can be extremely high. As a result, the biogeochemical signature of sediments near areas of active natural seepage is distinct: high concentrations of metabolic end products, such as dissolved inorganic carbon and hydrogen sulfide, abound, and often, high dissolved inorganic carbon concentrations result in the precipitation of authigenic carbonate minerals. We examined microbial processes and biogeochemical signatures at two natural seeps, Green Canyon 600 and Mississippi Canyon 118. Higher and more frequent seepage loci at the Green Canyon 600 site led to more widespread hotspots of elevated microbial activity and distinct geochemistry. However, rates of microbial activity were comparable at the two sites in areas of active hydrocarbon seepage. The microbial communities at the two sites were surprisingly different. The second group of sites was impacted by anthropogenic hydrocarbon discharges instead of natural seepage. One site, Oceanus 26, lies near the Deepwater Horizon/Macondo wellhead and was impacted by weathered oil sedimentation during the Macondo discharge. The second set of impacted sites, noted as Taylor Energy, lie near a sunken platform and compromised riser, which have together resulted in persistent hydrocarbon discharge to the adjacent oceanic system for more than 6 years. Rates of microbial activity in the upper sediments at Oceanus 26 were depressed relative to activity in the deeper layers, suggesting inhibition by the presence of weathered oil or an microbial community unable to weather the carbon available in the layer. At the Taylor energy site

  6. Anti-inflammation activities of mycosporine-like amino acids (MAAs) in response to UV radiation suggest potential anti-skin aging activity.

    PubMed

    Suh, Sung-Suk; Hwang, Jinik; Park, Mirye; Seo, Hyo Hyun; Kim, Hyoung-Shik; Lee, Jeong Hun; Moh, Sang Hyun; Lee, Taek-Kyun

    2014-10-14

    Certain photosynthetic marine organisms have evolved mechanisms to counteract UV-radiation by synthesizing UV-absorbing compounds, such as mycosporine-like amino acids (MAAs). In this study, MAAs were separated from the extracts of marine green alga Chlamydomonas hedleyi using HPLC and were identified as porphyra-334, shinorine, and mycosporine-glycine (mycosporine-Gly), based on their retention times and maximum absorption wavelengths. Furthermore, their structures were confirmed by triple quadrupole MS/MS. Their roles as UV-absorbing compounds were investigated in the human fibroblast cell line HaCaT by analyzing the expression levels of genes associated with antioxidant activity, inflammation, and skin aging in response to UV irradiation. The mycosporine-Gly extract, but not the other MAAs, had strong antioxidant activity in the 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Furthermore, treatment with mycosporine-Gly resulted in a significant decrease in COX-2 mRNA levels, which are typically increased in response to inflammation in the skin, in a concentration-dependent manner. Additionally, in the presence of MAAs, the UV-suppressed genes, procollagen C proteinase enhancer (PCOLCE) and elastin, which are related to skin aging, had increased expression levels equal to those in UV-mock treated cells. Interestingly, the increased expression of involucrin after UV exposure was suppressed by treatment with the MAAs mycosporine-Gly and shinorine, but not porphyra-334. This is the first report investigating the biological activities of microalgae-derived MAAs in human cells.

  7. Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis.

    PubMed

    Zalatan, Jesse G; Catrina, Irina; Mitchell, Rebecca; Grzyska, Piotr K; O'brien, Patrick J; Herschlag, Daniel; Hengge, Alvan C

    2007-08-01

    Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

  8. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  9. Possible active site of the sweet-tasting protein thaumatin.

    PubMed

    Slootstra, J W; De Geus, P; Haas, H; Verrips, C T; Meloen, R H

    1995-10-01

    Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

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

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

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

  11. 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

  12. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  13. A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

    PubMed

    O'Farrell, Norah; Kreiner, Michaela; Moore, Barry D; Parker, Marie-Claire

    2006-11-01

    We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).

  14. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  15. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  16. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  17. Estimation of protein function using template-based alignment of enzyme active sites

    PubMed Central

    2014-01-01

    Background The accumulation of protein structural data occurs more rapidly than it can be characterized by traditional laboratory means. This has motivated widespread efforts to predict enzyme function computationally. The most useful/accurate strategies employed to date are based on the detection of motifs in novel structures that correspond to a specific function. Functional residues are critical components of predictively useful motifs. We have implemented a novel method, to complement current approaches, which detects motifs solely on the basis of distance restraints between catalytic residues. Results ProMOL is a plugin for the PyMOL molecular graphics environment that can be used to create active site motifs for enzymes. A library of 181 active site motifs has been created with ProMOL, based on definitions published in the Catalytic Site Atlas (CSA). Searches with ProMOL produce better than 50% useful Enzyme Commission (EC) class suggestions for level 1 searches in EC classes 1, 4 and 5, and produce some useful results for other classes. 261 additional motifs automatically translated from Jonathan Barker’s JESS motif set [Bioinformatics 19:1644–1649, 2003] and a set of NMR motifs is under development. Alignments are evaluated by visual superposition, Levenshtein distance and root-mean-square deviation (RMSD) and are reasonably consistent with related search methods. Conclusion The ProMOL plugin for PyMOL provides ready access to template-based local alignments. Recent improvements to ProMOL, including the expanded motif library, RMSD calculations and output selection formatting, have greatly increased the program’s usability and speed, and have improved the way that the results are presented. PMID:24669788

  18. Proton nuclear Overhauser effect study of the heme active site structure of Coprinus macrorhizus peroxidase.

    PubMed

    Dugad, L B; Goff, H M

    1992-07-13

    Proton nuclear Overhauser effect and paramagnetic relaxation measurements have been used to define more extensively the heme active site structure of Coprinus macrorhizus peroxidase, CMP (previously known as Coprinus cinereus peroxidase), as the ferric low-spin cyanide ligated complex. The results are compared with other well-characterized peroxidase enzymes. The NMR spectrum of CMPCN shows changes in the paramagnetically shifted resonances as a function of time, suggesting a significant heme disorder for CMP. The presence of proximal and distal histidine amino acid residues are common to the heme environments of both CMPCN and HRPCN. However, the upfield distal arginine signals of HRPCN are not evident in the 1H-NMR spectra of CMPCN.

  19. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  20. Lavas from Active Boninite and Very Recent Basalt Eruptions at Two Submarine NE Lau Basin Sites

    NASA Astrophysics Data System (ADS)

    Rubin, K. H.; Embley, R. W.; Clague, D. A.; Resing, J. A.; Michael, P. J.; Keller, N. S.; Baker, E. T.

    2009-12-01

    preliminary 210Po-210Pb data on 5 NELSC lavas suggest the eruption occurred over at least a few months, with significant chemical heterogeneity (e.g., ~1 wt% MgO variation), and with highly enriched compositions (e.g., Th=3.3 ppm, Th/U >3.8). 210Po activity in 3 samples suggest a Nov 2008 eruption, consistent with interpretations from water column physical and chemical characteristics measured in Nov. 2008. 210Po in 2 other lavas suggest early 2009 and mid 2008 eruptions, respectively. Some young lavas at both volcanoes had native sulfur deposits on or within them, which has not been observed at recent submarine eruption sites on mid-ocean ridges or Loihi, but has been seen at NW Rota seamount (Mariana arc). Our goal is to define the age, duration, composition and magnitude of both NE Lau eruptions to help quantify magmatic, hydrothermal, and ecological impacts and geochemical signatures of interest to the US Ridge2000 and Margins programs, which partially supported the NE Lau response expedition. Geochemical characterization of samples is ongoing with shore-based collaborators.

  1. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  2. Catch-and-Hold Activation of Muscle Acetylcholine Receptors Having Transmitter Binding Site Mutations

    PubMed Central

    Purohit, Prasad; Bruhova, Iva; Gupta, Shaweta; Auerbach, Anthony

    2014-01-01

    Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process. PMID:24988344

  3. Sediment TCDD-EQs and EROD and MROD activities in Ranid frogs from agricultural and nonagricultural sites in Michigan (USA).

    PubMed

    Murphy, M B; Hecker, M; Coady, K K; Tompsett, A R; Jones, P D; Newsted, J L; Wong, H L; du Preez, L H; Solomon, K R; Carr, J A; Smith, E E; Kendall, R J; Van der Kraak, G; Giesy, J P

    2006-10-01

    In vitro studies have demonstrated atrazine-mediated induction of 7-ethoxyresorufin O-deethylase (EROD) activity. EROD is an enzyme active in the metabolism of many compounds, including many xenobiotics. These studies have suggested that atrazine may affect reproductive function by altering steroid metabolism. The goal of this study was to determine whether relationships could be detected between measured atrazine concentrations in surface waters and the liver-somatic index (LSI) and EROD and 7-methoxyresorufin O-deethylase (MROD) activities in the livers of ranid frogs. In addition, sediment dioxin toxic equivalents (TCDD-EQs) were determined using the H4IIE-luc cell bioassay. Adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana), and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south central Michigan in the summer of 2003. Atrazine concentrations at nonagricultural sites ranged from less than the limit of quantification (0.17 microg atrazine/L) to 0.23 microg atrazine/L and did not exceed 1.2 microg atrazine/L at agricultural sites. Sediment TCDD-EQs were measurable only at one agricultural site. Of the measured parameters, only LSI values in adult male frogs differed significantly between agricultural and nonagricultural sites, with greater values observed at agricultural sites. In green frogs, EROD and MROD activities were measurable in both adult and juvenile frogs and were similar among sites. Median EROD activities ranged from 13 to 21 pmol/min/mg protein in adult male green frogs and from 5 to 13 pmol/min/mg protein in adult female green frogs. Juvenile frogs had greater EROD and MROD activities than adult frogs. Bullfrogs and leopard frogs had greater activities than did green frogs. Atrazine concentrations were significantly and negatively correlated with MROD activity in adult male green frogs (Spearman R = -0.800). LSI and

  4. The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication*

    PubMed Central

    Chatterji, Udayan; Bobardt, Michael; Selvarajah, Suganya; Yang, Feng; Tang, Hengli; Sakamoto, Noayo; Vuagniaux, Gregoire; Parkinson, Tanya; Gallay, Philippe

    2009-01-01

    Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies. PMID:19380579

  5. Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II.

    PubMed

    Viktorovskaya, Olga V; Engel, Krysta L; French, Sarah L; Cui, Ping; Vandeventer, Paul J; Pavlovic, Emily M; Beyer, Ann L; Kaplan, Craig D; Schneider, David A

    2013-09-12

    Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.

  6. Screening breeding sites of the common toad (Bufo bufo) in England and Wales for evidence of endocrine disrupting activity.

    PubMed

    Pickford, Daniel B; Jones, Alexandra; Velez-Pelez, Alejandra; Orton, Frances; Iguchi, Taisen; Mitsui, Naoko; Tooi, Osamu

    2015-07-01

    Anuran amphibians are often present in agricultural landscapes and may therefore be exposed to chemicals in surface waters used for breeding. We used passive accumulation devices (SPMD and POCIS) to sample contaminants from nine breeding sites of the Common toad (Bufo bufo) across England and Wales, measuring endocrine activity of the extracts in a recombinant yeast androgen screen (YAS) and yeast estrogen screen (YES) and an in vitro vitellogenin induction screen in primary culture of Xenopus laevis hepatocytes. We also assessed hatching, growth, survival, and development in caged larvae in situ, and sampled metamorphs for gonadal histopathology. None of the SPMD extracts exhibited estrogen receptor or androgen receptor agonist activity, while POCIS extracts from two sites in west-central England exhibited concentration-dependent androgenic activity in the YAS. Three sites exhibited significant estrogenic activity in both the YES and the Xenopus hepatocyte. Hatching rates varied widely among sites, but there was no consistent correlation between hatching rate and intensity of agricultural activity, predicted concentrations of agrochemicals, or endocrine activity measured in YES/YAS assays. While a small number of intersex individuals were observed, their incidence could not be associated with predicted pesticide exposure or endocrine activitity measured in the in vitro screens. There were no significant differences in sex ratio, as determined by gonadal histomorphology among the study sites, and no significant correlation was observed between proportion of males and predicted exposure to agrochemicals. However, a negative correlation did become apparent in later sampling periods between proportion of males and estrogenic activity of the POCIS sample, as measured in the YES. Our results suggest that larval and adult amphibians may be exposed to endocrine disrupting chemicals in breeding ponds, albeit at low concentrations, and that chemical contaminants other than

  7. A new insight into the nature of seasonal variations in coordinate time series of GPS sites located near active faults

    NASA Astrophysics Data System (ADS)

    Trofimenko, Sergey V.; Bykov, Victor G.; Shestakov, Nikolay V.; Grib, Nikolay N.; Takahashi, Hiroaki

    2016-09-01

    This study provides new insights into the nature of seasonal variations in coordinate time series of GPS sites located near active faults and methods of their modeling. Monthly averaged coordinate time series were analyzed for several pairs of collocated GPS sites situated near the active fault intersection area, in close proximity to the central part of the northern boundary of the Amurian plate and the vicinity of the San Andreas Fault zone. It is concluded that the observed seasonal variations are best described by a breather function which is one of the solutions of the well-known sine-Gordon equation. The obtained results suggest that, in this case, the source of seasonal variations may be caused by the appearance of solitary strain waves in the fault intersection system, which may be qualitatively treated as standing waves of compression-extension of the geological medium. Based on statistical testing, the limits of applicability of the suggested model have been established.

  8. Improving Functional Annotation in the DRE-TIM Metallolyase Superfamily through Identification of Active Site Fingerprints.

    PubMed

    Kumar, Garima; Johnson, Jordyn L; Frantom, Patrick A

    2016-03-29

    Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C-C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth β-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the β3-5 and β4-7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the β3-5 and β4-7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the β3-5 and β4-7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup. PMID:26935545

  9. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  10. Spectroscopic definition of the copper active sites in mordenite: selective methane oxidation.

    PubMed

    Vanelderen, Pieter; Snyder, Benjamin E R; Tsai, Ming-Li; Hadt, Ryan G; Vancauwenbergh, Julie; Coussens, Olivier; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2015-05-20

    Two distinct [Cu-O-Cu](2+) sites with methane monooxygenase activity are identified in the zeolite Cu-MOR, emphasizing that this Cu-O-Cu active site geometry, having a ∠Cu-O-Cu ∼140°, is particularly formed and stabilized in zeolite topologies. Whereas in ZSM-5 a similar [Cu-O-Cu](2+) active site is located in the intersection of the two 10 membered rings, Cu-MOR provides two distinct local structures, situated in the 8 membered ring windows of the side pockets. Despite their structural similarity, as ascertained by electronic absorption and resonance Raman spectroscopy, the two Cu-O-Cu active sites in Cu-MOR clearly show different kinetic behaviors in selective methane oxidation. This difference in reactivity is too large to be ascribed to subtle differences in the ground states of the Cu-O-Cu sites, indicating the zeolite lattice tunes their reactivity through second-sphere effects. The MOR lattice is therefore functionally analogous to the active site pocket of a metalloenzyme, demonstrating that both the active site and its framework environment contribute to and direct reactivity in transition metal ion-zeolites.

  11. School Pharmacist/School Environmental Hygienic Activities at School Site.

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  12. 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.

  13. Genes Upregulated in Winter Wheat (Triticum aestivum L.) during Mild Freezing and Subsequent Thawing Suggest Sequential Activation of Multiple Response Mechanisms.

    PubMed

    Skinner, Daniel Z

    2015-01-01

    Exposing fully cold-acclimated wheat plants to a mild freeze-thaw cycle of -3 °C for 24h followed by +3 °C for 24 or 48 h results in dramatically improved tolerance of subsequent exposure to sub-freezing temperatures. Gene enrichment analysis of crown tissue from plants collected before or after the -3 °C freeze or after thawing at +3 °C for 24 or 48 h revealed that many biological processes and molecular functions were activated during the freeze-thaw cycle in an increasing cascade of responses such that over 150 processes or functions were significantly enhanced by the end of the 48 h, post-freeze thaw. Nearly 2,000 individual genes were upregulated more than 2-fold over the 72 h course of freezing and thawing, but more than 70% of these genes were upregulated during only one of the time periods examined, suggesting a series of genes and gene functions were involved in activation of the processes that led to enhanced freezing tolerance. This series of functions appeared to include extensive cell signaling, activation of stress response mechanisms and the phenylpropanoid biosynthetic pathway, extensive modification of secondary metabolites, and physical restructuring of cell membranes. By identifying plant lines that are especially able to activate these multiple mechanisms it may be possible to develop lines with enhanced winterhardiness.

  14. Microbial community changes along the active seepage site of one cold seep in the Red Sea

    PubMed Central

    Cao, Huiluo; Zhang, Weipeng; Wang, Yong; Qian, Pei-Yuan

    2015-01-01

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep. PMID:26284035

  15. Transition state stabilization by six arginines clustered in the active site of creatine kinase.

    PubMed

    Jourden, Michael J; Geiss, Paul R; Thomenius, Michael J; Horst, Lindsay A; Barty, Melissa M; Brym, Melissa J; Mulligan, Guy B; Almeida, Ryan M; Kersteen, Betsy A; Myers, Nichole R; Snider, Mark J; Borders, Charles L; Edmiston, Paul L

    2005-08-10

    Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.

  16. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.

    PubMed

    Kovaleva, Elena G; Rogers, Melanie S; Lipscomb, John D

    2015-09-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species.

  17. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine-200

    PubMed Central

    Kovaleva, Elena G.; Rogers, Melanie S.; Lipscomb, John D.

    2015-01-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures solved at 1.35 –1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species. PMID:26267790

  18. The influence of small-mammal burrowing activity on water storage at the Hanford Site

    SciTech Connect

    Landeen, D.S.

    1994-12-31

    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). 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.

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

    SciTech Connect

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

    2010-11-16

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

  20. Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology.

    PubMed

    Bellamine, Aouatef; Lepesheva, Galina I; Waterman, Michael R

    2004-11-01

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

  1. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120

    SciTech Connect

    Sheng, Jun; Charng, Yee-yung; Preiss, J.

    1996-03-05

    Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.

  2. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  3. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  4. 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...

  5. Activity of site-specific endonucleases on complexes of plasmid DNA with multiwalled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Veligura, A. A.; Shulitsky, B. G.; Asayonok, A. A.; Vaskovtsev, E. V.

    2016-08-01

    We have synthesized and investigated structural and functional properties of plasmid DNA complexes with multi-walled carbon nanotubes (MWCNTs) for detection of changes in structural state of the plasmid DNA at its recognition by site-specific endonuclease. It has been also established that the site-specific endonuclease is functionally active on the surface of MWCNTs.

  6. 77 FR 5560 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... project proposals on those leases) in identified Wind Energy Areas (WEAs) on the OCS offshore New Jersey... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the... site assessment plans (SAPs) on those leases. BOEM may issue one or more commercial wind energy...

  7. Hypnosis, suggestion, and suggestibility: an integrative model.

    PubMed

    Lynn, Steven Jay; Laurence, Jean-Roch; Kirsch, Irving

    2015-01-01

    This article elucidates an integrative model of hypnosis that integrates social, cultural, cognitive, and neurophysiological variables at play both in and out of hypnosis and considers their dynamic interaction as determinants of the multifaceted experience of hypnosis. The roles of these variables are examined in the induction and suggestion stages of hypnosis, including how they are related to the experience of involuntariness, one of the hallmarks of hypnosis. It is suggested that studies of the modification of hypnotic suggestibility; cognitive flexibility; response sets and expectancies; the default-mode network; and the search for the neurophysiological correlates of hypnosis, more broadly, in conjunction with research on social psychological variables, hold much promise to further understanding of hypnosis.

  8. Structure of HIV-1 Reverse Transcriptase with the Inhibitor -thujaplicinol Bound at the RNase H Active Site

    SciTech Connect

    Himmel, D.; Maegley, K; Pauly, T; Bauman, J; Das, K; Dharia, C; Clark, Jr., A; Ryan, K; Hickey, M; et al.

    2009-01-01

    Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, such as RNH, should be effective against all of the current drug-resistant variants. Here, we present 2.80 {angstrom} and 2.04 {angstrom} resolution crystal structures of an RNH inhibitor, {beta}-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. {beta}-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that {beta}-thujaplicinol is a slow-binding RNH inhibitor with noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

  9. Suggested Involvement of PP1/PP2A Activity and De Novo Gene Expression in Anhydrobiotic Survival in a Tardigrade, Hypsibius dujardini, by Chemical Genetic Approach.

    PubMed

    Kondo, Koyuki; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade, Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades.

  10. Suggested Involvement of PP1/PP2A Activity and De Novo Gene Expression in Anhydrobiotic Survival in a Tardigrade, Hypsibius dujardini, by Chemical Genetic Approach

    PubMed Central

    Kondo, Koyuki; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade, Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades. PMID:26690982

  11. The Role of an Active Site Mg2+ in HDV Ribozyme Self-Cleavage: Insights from QM/MM Calculations

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

    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 Mg2+ ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with distinct position and coordination of the catalytically important active site Mg2+ ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal/mol, indicating that the specific position of the Mg2+ 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 estimated the pKa of the U-1(2′-OH) group to range from 8.8 to 11.2, suggesting that the pKa 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 Mg2+ ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg2+ ion, facilitates deprotonation and activation of the 2′-OH nucleophile. PMID:25412464

  12. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  13. 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.

  14. 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.

  15. 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

  16. Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein-RNA interactions.

    PubMed

    Maravić, Gordana; Bujnicki, Janusz M; Feder, Marcin; Pongor, Sándor; Flögel, Mirna

    2003-08-15

    The Erm family of adenine-N(6) methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Hence, these proteins are important potential drug targets. Despite the availability of the NMR and crystal structures of two members of the family (ErmAM and ErmC', respectively) and extensive studies on the RNA substrate, the substrate-binding site and the amino acids involved in RNA recognition by the Erm MTases remain unknown. It has been proposed that the small C-terminal domain functions as a target-binding module, but this prediction has not been tested experimentally. We have undertaken structure-based mutational analysis of 13 charged or polar residues located on the predicted rRNA-binding surface of ErmC' with the aim to identify the area of protein-RNA interactions. The results of in vivo and in vitro analyses of mutant protein suggest that the key RNA-binding residues are located not in the small domain, but in the large catalytic domain, facing the cleft between the two domains. Based on the mutagenesis data, a preliminary three-dimensional model of ErmC' complexed with the minimal substrate was constructed. The identification of the RNA-binding site of ErmC' may be useful for structure-based design of novel drugs that do not necessarily bind to the cofactor-binding site common to many S-adenosyl-L- methionine-dependent MTases, but specifically block the substrate-binding site of MTases from the Erm family. PMID:12907737

  17. Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein-RNA interactions.

    PubMed

    Maravić, Gordana; Bujnicki, Janusz M; Feder, Marcin; Pongor, Sándor; Flögel, Mirna

    2003-08-15

    The Erm family of adenine-N(6) methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Hence, these proteins are important potential drug targets. Despite the availability of the NMR and crystal structures of two members of the family (ErmAM and ErmC', respectively) and extensive studies on the RNA substrate, the substrate-binding site and the amino acids involved in RNA recognition by the Erm MTases remain unknown. It has been proposed that the small C-terminal domain functions as a target-binding module, but this prediction has not been tested experimentally. We have undertaken structure-based mutational analysis of 13 charged or polar residues located on the predicted rRNA-binding surface of ErmC' with the aim to identify the area of protein-RNA interactions. The results of in vivo and in vitro analyses of mutant protein suggest that the key RNA-binding residues are located not in the small domain, but in the large catalytic domain, facing the cleft between the two domains. Based on the mutagenesis data, a preliminary three-dimensional model of ErmC' complexed with the minimal substrate was constructed. The identification of the RNA-binding site of ErmC' may be useful for structure-based design of novel drugs that do not necessarily bind to the cofactor-binding site common to many S-adenosyl-L- methionine-dependent MTases, but specifically block the substrate-binding site of MTases from the Erm family.

  18. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.

    PubMed

    Daudé, David; Topham, Christopher M; Remaud-Siméon, Magali; André, Isabelle

    2013-12-01

    The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

  19. Probing Binding Sites and Mechanisms of Action of an IKs Activator by Computations and Experiments

    PubMed Central

    Xu, Yu; Wang, Yuhong; Zhang, Mei; Jiang, Min; Rosenhouse-Dantsker, Avia; Wassenaar, Tsjerk; Tseng, Gea-Ny

    2015-01-01

    The slow delayed rectifier (IKs) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. IKs activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance IKs amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4–S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K+ ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates IKs by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect. PMID:25564853

  20. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

  1. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  2. Immunolocalization of Anti-Hsf1 to the Acetabular Glands of Infectious Schistosomes Suggests a Non-Transcriptional Function for This Transcriptional Activator

    PubMed Central

    Knudsen, Giselle M.; Jolly, Emmitt R.

    2014-01-01

    Schistosomiasis is a chronically debilitating disease caused by parasitic worms of the genus Schistosoma, and it is a global problem affecting over 240 million people. Little is known about the regulatory proteins and mechanisms that control schistosome host invasion, gene expression, and development. Schistosome larvae, cercariae, are transiently free-swimming organisms and infectious to man. Cercariae penetrate human host skin directly using proteases that degrade skin connective tissue. These proteases are secreted from anucleate acetabular glands that contain many proteins, including heat shock proteins. Heat shock transcription factors are strongly conserved activators that play crucial roles in the maintenance of cell homeostasis by transcriptionally regulating heat shock protein expression. In this study, we clone and characterize the schistosome Heat shock factor 1 gene (SmHSF1). We verify its ability to activate transcription using a modified yeast one-hybrid system, and we show that it can bind to the heat shock binding element (HSE) consensus DNA sequence. Our quantitative RT-PCR analysis shows that SmHSF1 is expressed throughout several life-cycle stages from sporocyst to adult worm. Interestingly, using immunohistochemistry, a polyclonal antibody raised against an Hsf1-peptide demonstrates a novel localization for this conserved, stress-modulating activator. Our analysis suggests that schistosome Heat shock factor 1 may be localized to the acetabular glands of infective cercariae. PMID:25078989

  3. Psychosocial predictors of decay in healthy eating and physical activity improvements in obese women regaining lost weight: translation of behavioral theory into treatment suggestions.

    PubMed

    Annesi, James J

    2016-06-01

    Regain of lost weight is a universal problem for behavioral treatments. An increased understanding of theory-based psychosocial predictors of decay in behavioral correlates of weight loss might improve treatments. Data were derived from a previous weight loss investigation of 110 women with obesity. A subsample from the experimental treatment who lost ≥3 % body weight and regained at least one third of that over 24 months (N = 36) was assessed. During months 6 through 24, there were unfavorable changes in behavioral (fruit/vegetable and sweet intake; physical activity) and psychosocial variables. Mood change predicted change in fruit/vegetable and sweet intake, with emotional eating change mediating the latter relationship. Change in self-regulation predicted changes in sweet and fruit/vegetable intake and physical activity, with self-efficacy mediating the self-regulation-fruit/vegetable intake and self-regulation-physical activity relationships. Findings suggest that after treatment-induced weight loss, addressing indicated theory-based psychosocial variables might mitigate decay in behavioral predictors of healthier weight. PMID:27052217

  4. Clonality Analysis of Immunoglobulin Gene Rearrangement by Next-Generation Sequencing in Endemic Burkitt Lymphoma Suggests Antigen Drive Activation of BCR as Opposed to Sporadic Burkitt Lymphoma

    PubMed Central

    Amato, Teresa; Abate, Francesco; Piccaluga, Pierpaolo; Iacono, Michele; Fallerini, Chiara; Renieri, Alessandra; De Falco, Giulia; Ambrosio, Maria Raffaella; Mourmouras, Vaselious; Ogwang, Martin; Calbi, Valeria; Rabadan, Roul; Hummel, Michael; Pileri, Stefano; Bellan, Cristiana

    2016-01-01

    Objectives: Recent studies using next-generation sequencing (NGS) analysis disclosed the importance of the intrinsic activation of the B-cell receptor (BCR) pathway in the pathogenesis of sporadic Burkitt lymphoma (sBL) due to mutations of TCF3/ID3 genes. Since no definitive data are available on the genetic landscape of endemic Burkitt (eBL), we first assessed the mutation frequency of TCF3/ID3 in eBL compared with sBL and subsequently the somatic hypermutation status of the BCR to answer whether an extrinsic activation of BCR signaling could also be demonstrated in Burkitt lymphoma. Methods: We assessed the mutations of TCF3/ID3 by RNAseq and the BCR status by NGS analysis of the immunoglobulin genes (IGs). Results: We detected mutations of TCF3/ID3 in about 30% of the eBL cases. This rate is significantly lower than that detected in sBL (64%). The NGS analysis of IGs revealed intraclonal diversity, suggesting an active targeted somatic hypermutation process in eBL compared with sBL. Conclusions: These findings support the view that the antigenic pressure plays a key role in the pathogenetic pathways of eBL, which may be partially distinct from those driving sBL development. PMID:26712879

  5. Nuclear waste: Status of DOE`s nuclear waste site characterization activities

    SciTech Connect

    1987-12-31

    Three potential nuclear waste repository sites have been selected to carry out characterization activities-the detailed geological testing to determine the suitability of each site as a repository. The sites are Hanford in south-central Washington State, Yucca Mountain in southern Nevada, and Deaf Smith in the Texas Panhandle. Two key issues affecting the total program are the estimations of the site characterization completion data and costs and DOE`s relationship with the Nuclear Regulatory Commission which has been limited and its relations with affected states and Indian tribes which continue to be difficult.

  6. XAFS Study of the Photo-Active Site of Mo/MCM-41

    NASA Astrophysics Data System (ADS)

    Miyamoto, Daisuke; Ichikuni, Nobuyuki; Shimazu, Shogo

    2007-02-01

    An Mo/MCM-41 catalyst was prepared and used for study of propene and 1-butene photo-metathesis reactions. XAFS analysis revealed that hydrogen reduction leads to a decreased role for the Mo=O site. The Mo-O site plays an important role for the olefin photo-metathesis reaction on the H2 reduced Mo/MCM-41. From EXAFS analysis, the active site of photo-metathesis reaction is the Mo=O part for oxidized Mo/MCM-41, whereas it is the Mo-O site for reduced Mo/MCM-41.

  7. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  8. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  9. Coordination number of zinc ions in the phosphotriesterase active site by molecular dynamics and quantum mechanics.

    PubMed

    Koca, Jaroslav; Zhan, Chang-Guo; Rittenhouse, Robert C; Ornstein, Rick L

    2003-02-01

    We have run several molecular dynamics (MD) simulations on zinc-containing phosphotriesterase (PTE) with two bound substrates, sarin and paraoxon, and with the substrate analog diethyl 4-methylbenzylphosphonate. A standard nonbonded model was employed to treat the zinc ions with the commonly used charge of +2. In all the trajectories, we observed a tightly bound water (TBW) molecule in the active site that was coordinated to the less buried zinc ion. The phosphoryl oxygen of the substrate/inhibitor was found to be coordinated to the same zinc ion so that, considering all ligands, the less buried zinc was hexa-coordinated. The hexa-coordination of this zinc ion was not seen in the deposited X-ray pdb files for PTE. Several additional MD simulations were then performed using different charges (+1, +1.5) on the zinc ions, along with ab initio and density functional theory (DFT) calculations, to evaluate the following possibilities: the crystal diffraction data were not correctly interpreted; the hexa-coordinated zinc ion in PTE is only present in solution and not in the crystal; and the hexa-coordinated zinc ion in PTE is an artifact of the force field used. A charge of +1.5 leads to a coordination number (CN) of 5 on both zinc ions, which is consistent with the results from ab initio and DFT calculations and with the latest high resolution X-ray crystal structure. The commonly used charge of +2 produces a CN of 6 on the less buried zinc. The CN on the more buried zinc ion is 5 when the substrate/inhibitor is present in the simulation, and increases to 6 when the substrate/inhibitor is removed prior to the simulation. The results of both of the MD and quantum mechanical calculations lead to the conclusion that the zinc ions in the PTE active site are both penta-coordinated, and that the MD simulations performed with the charge of +2 overestimate the CN of the zinc ions in the PTE active site. The overall protein structures in the simulations remain unaffected by the

  10. Transcriptional activation through ETS domain binding sites in the cytochrome c oxidase subunit IV gene

    SciTech Connect

    Virbasius, J.V.; Scarpulla, R.C. )

    1991-11-01

    A mutational analysis of the rat cytochrome c oxidase subunit IV (RCO4) promoter region revealed the presence of a major control element consisting of a tandemly repeated pair of binding sites for a nuclear factor from HeLa cells. This factor was designated NRF-2 (nuclear respiratory factor 2) because a functional recognition site was also found in the human ATP synthase {beta}-subunit gene. Deletion or site-directed point mutations of the NRF-2 binding sites in the RCO4 promoter resulted in substantial loss of transcriptional activity, and synthetic oligomers of the NRF-2 binding sites from both genes stimulated a heterologous promoter when cloned in cis. NRF-2 binding a transcriptional activation required a purine-rich core sequence, GGAA. This motif is characteristic of the recognition site for a family of activators referred to as ETS domain proteins because of the similarity within their DNA-binding domains to the ets-1 proto-oncogene product. NRF-2 recognized an authentic Ets-1 site within the Moloney murine sarcoma virus long terminal repeat, and this site was able to compete for NRF-2 binding to the RCO4 promoter sequence. However, in contrast to Ets-1, which appears to be exclusive to lymphoid tissues, NRF-2 has the broad tissue distribution expected of a regulator of respiratory chain expression.

  11. Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase.

    PubMed

    Fafarman, Aaron T; Sigala, Paul A; Schwans, Jason P; Fenn, Timothy D; Herschlag, Daniel; Boxer, Steven G

    2012-02-01

    Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV/cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK(a) prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK(a) near 7 was included. UV-absorbance and (13)C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis.

  12. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  13. Ligand-dependent dynamics of the active-site lid in bacterial dimethylarginine dimethylaminohydrolase.

    PubMed

    Rasheed, Masooma; Richter, Christine; Chisty, Liisa T; Kirkpatrick, John; Blackledge, Martin; Webb, Martin R; Driscoll, Paul C

    2014-02-18

    The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed. PMID:24484052

  14. 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

  15. Active and diverse rainwater bacteria collected at an inland site in spring and summer 2011

    NASA Astrophysics Data System (ADS)

    Cho, Byung Cheol; Jang, Gwang Il

    2014-09-01

    Rainwater is an important natural resource and utilized for various beneficial purposes. However, information on prokaryotes in rainwater is limited. Rainwater samples were collected during three heavy rain events at a suburban site in Seoul in April, May, and July 2011. The highest bacterial abundance (BA) in rainwater was observed in April when airborne bacteria had also been abundant the day before rainwater collection. ATP content in bacterial fraction of the rainwater suggested that the rainwater bacteria were metabolically active. Bacterial community compositions (BCCs) of rainwater samples, analyzed by using 16S rRNA gene-based pyrosequencing, differed considerably among the three rain events. Rainwater bacteria showed potentials of fast growth and drastic shift after incubation in BCCs from fresh rainwater at broad taxonomic levels and the dominant operational taxonomic units (OTUs) level. Presumable marine bacterial OTUs which formed a robust clade with marine bacteria Lacinutrix spp. were at high concentrations in rainwater in April, likely reflecting origin from saline environments. Most of the Flavobacteria sequences unusually high in April rainwater seemed to have marine origins. Further, spore-forming euryhaline marine Firmicutes were isolated from rainwater samples, suggesting possible dispersal of some marine bacteria via rain. A potential human pathogen and Escherichia coli-like sequences were detected in rainwater samples, calling for the need for assessment of health risks of collected rainwater.

  16. Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II

    PubMed Central

    Viktorovskaya, Olga V.; Engel, Krysta L.; French, Sarah L.; Cui, Ping; Vandeventer, Paul J.; Pavlovic, Emily M.; Beyer, Ann L.; Kaplan, Craig D.; Schneider, David A.

    2013-01-01

    SUMMARY Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss-of-function in Pol I [Pol II: rpb1- E1103G; Pol I: rpa190-E1224G]. Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase function, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps. PMID:23994471

  17. Sugar binding effects on the enzymatic reaction and conformation near the active site of pokeweed antiviral protein revealed by fluorescence spectroscopy.

    PubMed

    Nakashima, Hiromichi; Fukunaga, Yukihiro; Ueno, Ryosuke; Nishimoto, Etsuko

    2014-05-01

    In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

  18. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-09-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

  19. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  20. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.

    PubMed

    Hirschi, Alexander; Cecchini, Matthew; Steinhardt, Rachel C; Schamber, Michael R; Dick, Frederick A; Rubin, Seth M

    2010-09-01

    The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking. PMID:20694007

  1. Selectivity loss of Pt/CeO{sub 2} PROX catalysts at low CO concentrations: mechanism and active site study.

    SciTech Connect

    Polster, C. S.; Zhang, R.; Cyb, M. T.; Miller, J. T.; Baertsch, C. D.

    2010-07-01

    CO and H{sub 2} oxidation were studied over a series of Pt/CeO{sub 2} catalysts with differing Pt loadings and dispersions. Kinetic rate analysis confirms the presence of dual Langmuir-Hinshelwood (L-H) and Mars and van Krevelen (M-vK) pathways and is used to explain the loss in CO oxidation selectivity at low CO concentrations. In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) shows the strong CO coverage dependence on both CO and O{sub 2} concentrations and explains the transition from L-H to M-vK reaction character. Redox site measurements are performed on Pt/CeO{sub 2} catalysts by anaerobic titrations under conditions where the M-vK pathway dominates the reaction rate. Similar redox site densities per interfacial Pt atom suggest that interfacial Pt-O-Ce sites are responsible for M-vK redox activity.

  2. Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters.

    PubMed Central

    Cromlish, W A; Abmayr, S M; Workman, J L; Horikoshi, M; Roeder, R G

    1989-01-01

    In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements. Images PMID:2539489

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

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  4. Preseismic velocity changes observed from active source monitoring at the Parkfield SAFOD drill site.

    PubMed

    Niu, Fenglin; Silver, Paul G; Daley, Thomas M; Cheng, Xin; Majer, Ernest L

    2008-07-10

    Measuring stress changes within seismically active fault zones has been a long-sought goal of seismology. One approach is to exploit the stress dependence of seismic wave velocity, and we have investigated this in an active source cross-well experiment at the San Andreas Fault Observatory at Depth (SAFOD) drill site. Here we show that stress changes are indeed measurable using this technique. Over a two-month period, we observed an excellent anti-correlation between changes in the time required for a shear 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 travel-time data that are coincident with two earthquakes that are among those predicted to produce the largest coseismic stress changes at SAFOD. 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.

  5. Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

    SciTech Connect

    Igarashi, Y.; McFadden, B.A.; el-Gul, T.

    1985-07-16

    (TH) Diethyl pyrocarbonate was synthesized from (TH) ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM MgS , and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with earlier experiments, suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources.

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

    PubMed

    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

  7. Spacing between GT-1 binding sites within a light-responsive element is critical for transcriptional activity.

    PubMed Central

    Gilmartin, P M; Chua, N H

    1990-01-01

    Dissection of the light-responsive element (LRE) located between -166 and -50 of rbcS-3A from pea has revealed critical spacing requirements between the two GT-1 binding sites for light-responsive transcription. An increase in spacing between the two sites by as little as 2 bp reduces dramatically the rbcS-3A transcript levels in vivo. Mutation of the 10 bp between the binding sites leads to slightly lower transcript levels, as do deletions of either 3 bp or 8 bp. Deletions of 5 bp or 7 bp from between the GT-1 binding sites do not affect rbcS-3A transcript levels; however, a deletion of 10 bp virtually abolishes the activity of this element. These spacing changes within the light-responsive element similarly affect transcription of a divergently oriented and truncated nopaline synthase promoter. Most spacing changes between the two GT-1 binding sites, however, do not impair the binding of GT-1 to this element in vitro. Together with previous observations, these results suggest that the nuclear factor GT-1 may interact with the binding sites in either a productive or nonproductive manner and that GT-1 binding is necessary but not sufficient for light-responsive transcription. We also discuss our results in relation to the observed spacing of similar sequence elements present within other light-responsive promoters. PMID:2152170

  8. Mechanistic pathways of mercury removal from the organomercurial lyase active site.

    PubMed

    Silva, Pedro J; Rodrigues, Viviana

    2015-01-01

    protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket. PMID:26246970

  9. Mechanistic pathways of mercury removal from the organomercurial lyase active site

    PubMed Central

    Rodrigues, Viviana

    2015-01-01

    protonation by solvent-provided H3O+. Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket. PMID:26246970

  10. Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

    PubMed Central

    Soh, Y; Song, B J; Jeng, J; Kallarakal, A T

    1998-01-01

    It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity. PMID:9657977

  11. A 7-phenyl substituted triazolopyridazine has inverse agonist activity at the benzodiazepine receptor site.

    PubMed Central

    Biziere, K.; Bourguignon, J. J.; Chambon, J. P.; Heaulme, M.; Perio, A.; Tebib, S.; Wermuth, C. G.

    1987-01-01

    To investigate further the structural requirements for benzodiazepine (BZD) receptor ligands, we synthesized SR 95195, [7-phenyl-3-methyl-1,2,4-triazolo-(4,3-b) pyridazine], a positional isomer of the 6-phenyl-triazolo-pyridazines, which were the first non-BZD derivatives to exhibit high affinity for the BZD receptor and BZD-like activity in vivo. In vitro, SR 95195 displaced specifically bound [3H]-flunitrazepam from rat cerebellar and hippocampal membranes with respective IC50 values of 4 and 8 microM. In vivo, SR 95195 lacked BZD-like activity. At high doses SR 95195 induced clonic seizures in mice (threshold convulsant dose: 150 mg kg-1; CD50: 160 mg kg-1 i.p.) which were antagonized by Ro 15-1788. At non-convulsant doses (25 mg kg-1 i.p. and 100 mg kg-1 i.p.) SR 95195 significantly decreased punished responding in an operant conflict procedure in the rat, suggesting SR 95195 has intrinsic anxiogenic activity. SR 95195, in mice, reversed the anticonvulsant and myorelaxant actions of diazepam 3 mg kg-1, orally (respective ED50 values: 45 mg kg-1 i.p. and 44 mg kg-1 i.p.). In an operant-conflict test in rats, SR 95195 at non-anxiogenic doses, antagonized the disinhibitory action of diazepam 4 mg kg-1, i.p. (ED50: 8.6 mg kg-1, i.p.), but not that of pentobarbitone 15 mg kg-1, i.p. It is concluded that SR 95195 has the pharmacological profile of an inverse BZD agonist and that displacing the phenyl from the 6- to the 7-position in the triazolopyridazine series causes a shift from agonist to inverse agonist type activity at the BZD receptor site. PMID:3028557

  12. 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

  13. Non-photochemical quenching and xanthophyll cycle activities in six green algal species suggest mechanistic differences in the process of excess energy dissipation.

    PubMed

    Quaas, Theresa; Berteotti, Silvia; Ballottari, Matteo; Flieger, Kerstin; Bassi, Roberto; Wilhelm, Christian; Goss, Reimund

    2015-01-01

    In the present study the non-photochemical quenching (NPQ) of four biofilm-forming and two planktonic green algae was investigated by fluorescence measurements, determinations of the light-driven proton gradient and determination of the violaxanthin cycle activity by pigment analysis. It was observed that, despite the common need for efficient photoprotection, the structural basis of NPQ was heterogeneous in the different species. Three species, namely Chlorella saccharophila, Chlorella vulgaris and Bracteacoccus minor, exhibited a zeaxanthin-dependent NPQ, while in the three other species, Tetracystis aeria, Pedinomonas minor and Chlamydomonas reinhardtii violaxanthin de-epoxidation was absent or unrelated to the establishment of NPQ. Acclimation of the algae to high light conditions induced an increase of the NPQ activity, suggesting that a significant part of the overall NPQ was rather inducible than constitutively present in the green algae. Comparing the differences in the NPQ mechanisms with the phylogenetic position of the six algal species led to the conclusion that the NPQ heterogeneity observed in the present study was not related to the phylogeny of the algae but to the environmental selection pressure. Finally, the difference in the NPQ mechanisms in the different species is discussed within the frame of the current NPQ models.

  14. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

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

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  15. Characterizations of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

    SciTech Connect

    Chai,S.; Ju, T.; Dang, M.; Goldsmith, R.; Maroney, M.; Pochapsky, T.

    2008-01-01

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1, 2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2+ metal ion bound in the active site. The Ni2+-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2+-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates metal in vivo but

  16. Characterization of Metal Binding in the Active Sites of acireductone dioxygenase Isoforms from Klebsiella ATCC 8724

    SciTech Connect

    S Chai; T Ju; M Dang; R Goldsmith; M Maroney; T Pochapsky

    2011-12-31

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M{sup 2+} metal ion bound in the active site. The Ni{sup 2+}-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe{sup 2+}-bound FeARD catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates

  17. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  18. 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

  19. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 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...

  20. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 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...

  1. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 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...

  2. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the... Activities on the Atlantic OCS Offshore RI and MA'' to: Program Manager, Office of Renewable Energy...

  3. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

  4. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO.

  5. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  6. Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge.

    PubMed

    Tara, S; Elcock, A H; Kirchhoff, P D; Briggs, J M; Radic, Z; Taylor, P; McCammon, J A

    1998-12-01

    It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.

  7. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

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

    PubMed Central

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

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  9. 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.

  10. Structure of Arabidopsis thaliana 5-methylthioribose Kinase Reveals a More Occluded Active Site Than its Bacterial Homolog

    SciTech Connect

    Ku,S.; Cornell, K.; Howell, P.

    2007-01-01

    Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATP?S and MTR has been determined at 1.9 Angstroms resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATP?S analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

  11. Open to Suggestion.

    ERIC Educational Resources Information Center

    Journal of Reading, 1987

    1987-01-01

    Offers (1) suggestions for improving college students' study skills; (2) a system for keeping track of parent, teacher, and community contacts; (3) suggestions for motivating students using tic tac toe; (4) suggestions for using etymology to improve word retention; (5) a word search grid; and (6) suggestions for using postcards in remedial reading…

  12. Unusual Extra Space at the Active Site and High Activity for Acetylated Hydroxyproline of Prolyl Aminopeptidase from Serratia marcescens

    PubMed Central

    Nakajima, Yoshitaka; Ito, Kiyoshi; Sakata, Makoto; Xu, Yue; Nakashima, Kanako; Matsubara, Futoshi; Hatakeyama, Susumi; Yoshimoto, Tadashi

    2006-01-01

    The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 Å resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-βNA (4-acetyloxyproline β-naphthylamide) was a better substrate than Pro-βNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria. PMID:16452443

  13. The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans.

    PubMed

    Panozzo, C; Capuano, V; Fillinger, S; Felenbok, B

    1997-09-01

    The alcA gene which is part of the recently identified ethanol regulon, is one of the most strongly inducible genes in Aspergillus nidulans. Its transcriptional activation is mediated by the AlcR transactivator which contains a DNA-binding domain belonging to the C6 zinc binuclear cluster family. AlcR differs from the other members of this family by several features, the most striking characteristic being its binding to both symmetric and asymmetric DNA sites with the same apparent affinity. However, AlcR is also able to bind to a single site with high affinity, suggesting that unlike the other C6 proteins, AlcR binds as a monomer. In this report, we show that AlcR targets, to be functional in vivo, have to be organized as inverted or direct repeats. In addition, we show a strong synergistic activation of alcA transcription in which the number and the position of the AlcR-binding sites are crucial. The fact that the AlcR unit for in vitro binding is a single site whereas the in vivo functional unit is a repeat opens the question of the mechanism of the strong alcA transactivation. These results show that AlcR displays both in vitro and in vivo a new range of binding specificity and provides a novel example in the C6 zinc cluster protein family.

  14. The complexities of measuring access to parks and physical activity sites in New York City: a quantitative and qualitative approach

    PubMed Central

    Maroko, Andrew R; Maantay, Juliana A; Sohler, Nancy L; Grady, Kristen L; Arno, Peter S

    2009-01-01

    Background Proximity to parks and physical activity sites has been linked to an increase in active behaviors, and positive impacts on health outcomes such as lower rates of cardiovascular disease, diabetes, and obesity. Since populations with a low socio-economic status as well as racial and ethnic minorities tend to experience worse health outcomes in the USA, access to parks and physical activity sites may be an environmental justice issue. Geographic Information systems were used to conduct quantitative and qualitative analyses of park accessibility in New York City, which included kernel density estimation, ordinary least squares (global) regression, geographically weighted (local) regression, and longitudinal case studies, consisting of field work and archival research. Accessibility was measured by both density of park acreage and density of physical activity sites. Independent variables included percent non-Hispanic black, percent Hispanic, percent below poverty, percent of adults without high school diploma, percent with limited English-speaking ability, and population density. Results The ordinary least squares linear regression found weak relationships in both the park acreage density and the physical activity site density models (Ra2 = .11 and .23, respectively; AIC = 7162 and 3529, respectively). Geographically weighted regression, however, suggested spatial non-stationarity in both models, indicating disparities in accessibility that vary over space with respect to magnitude and directionality of the relationships (AIC = 2014 and -1241, respectively). The qualitative analysis supported the findings of the local regression, confirming that although there is a geographically inequitable distribution of park space and physical activity sites, it is not globally predicted by race, ethnicity, or socio-economic status. Conclusion The combination of quantitative and qualitative analyses demonstrated the complexity of the issues around racial and ethnic

  15. Single-agent lenalidomide in relapsed/refractory mantle cell lymphoma: results from a UK phase II study suggest activity and possible gender differences.

    PubMed

    Eve, Heather E; Carey, Sean; Richardson, Sarah J; Heise, Carla C; Mamidipudi, Vidya; Shi, Tao; Radford, John A; Auer, Rebecca L; Bullard, Sheila H; Rule, Simon A J

    2012-10-01

    We present data from a phase II study investigating a novel treatment strategy for relapsed/refractory mantle cell lymphoma (MCL). Twenty-six patients received lenalidomide 25 mg/d (days 1-21 of a 28-d cycle) for up to 6 cycles followed by low-dose maintenance lenalidomide (15 mg) in responding patients. Eight patients achieved complete or partial response to give an overall response rate of 31% with median response duration of 22·2 months [95% confidence interval (CI) 0·0-53·6] and median progression-free survival (PFS) of 3·9 months (95% CI 0·0-11·1). An additional six patients (23%) achieved stable disease. Eleven patients received maintenance with median PFS of 14·6 months (95% CI 7·3-21·9). Correlative studies showed that peripheral T and Natural Killer (NK) cells increased in responding patients by 40-60% over the first 6 cycles with an initial dip in NK cells suggestive of tumour infiltration. Peripheral regulatory T cells were increased in MCL patients (P = 0·001) and expanded further following lenalidomide. Sequential plasma analysis showed increased IL12 p40 and IL7 alongside decreased MMP9, IL10, and adiponectin. Finally, a significant correlation (P = 0·02) between gender and response suggested that female MCL patients were more sensitive to lenalidomide than males. In summary, we confirm the activity, safety and immunomodulatory properties of lenalidomide in MCL and highlight its potential as a low-dose maintenance agent.

  16. 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.

  17. Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila.

    PubMed

    Gorrell, Andrea; Lawrence, Sarah H; Ferry, James G

    2005-03-18

    Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate. PMID:15647264

  18. Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands.

    PubMed

    Nolte, H J; Rosenberry, T L; Neumann, E

    1980-08-01

    The reaction of the specific fluorescent cationic ligand N-methylacridinium with the active site of 11S acetylcholinesterase from electric eel was monitored by temperature-jump relaxation kinetics at a variety of ionic strengths. The ionic strength dependence of the bimolecular association rate constant is analyzed with a Brønsted-Debye-Hückel expression and leads to estimates of the association rate constant at zero ionic strength of K120 = 1.1 X 10(10) M-1 S-1 at 25 degrees C and the net charge number of the enzyme active site of ZE = -6.3. The ionic strength dependence of the second-order hydrolysis rate constant kcat/Kapp for acetylthiocholine under steady-state conditions is also very pronounced and indicates a value of ZE = -9. Thus, a large effective negative charge on the enzyme active site appears to be a general characteristic of its interaction with cationic ligands. The ionic strength dependence of Kcat/Kapp is identical with that of sodium chloride, sodium phosphate, and sodium citrate, thus ruling out any possibility that the phenomena arise from a specific, partially competitive binding of Na+ to the enzyme active site. Substitution of the calculated electrostatic parameters into theoretical equations indicates that the most significant effect of these ZE values is a 2-3 order of magnitude reduction in the rate constant for dissociation of the initial ligand-enzyme encounter complex; this decrease renders the bimolecular reaction diffusion controlled. The high value of k120 and the space requirements of six to nine charged groups suggest that regions of the enzyme surface area larger than the catalytic sites themselves are effective in trapping cationic ligands.

  19. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    SciTech Connect

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M.

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  20. Activation of protein phosphatase 1 by a small molecule designed to bind to the enzyme's regulatory site.

    PubMed

    Tappan, Erin; Chamberlin, A Richard

    2008-02-01

    The activity of protein phosphatase 1 (PP1), a serine-threonine phosphatase that participates ubiquitously in cellular signaling, is controlled by a wide variety of regulatory proteins that interact with PP1 at an allosteric regulatory site that recognizes a "loose" consensus sequence (usually designated as RVXF) found in all such regulatory proteins. Peptides containing the regulatory consensus sequence have been found to recapitulate the binding and PP1 activity modulation of the regulatory proteins, suggesting that it might be possible to design small-molecule surrogates that activate PP1 rather than inhibiting it. This prospect constitutes a largely unexplored way of controlling signaling pathways that could be functionally complementary to the much more extensively explored stratagem of kinase inhibition. Based on these principles, we have designed a microcystin analog that activates PP1. PMID:18291321

  1. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  2. Genome wide survey and molecular modeling of hypothetical proteins containing 2Fe-2S and FMN binding domains suggests Rieske Dioxygenase Activity highlighting their potential roles in bioremediation.

    PubMed

    Sathyanarayanan, Nitish; Nagendra, Holenarsipur Gundurao

    2014-01-01

    'Conserved hypothetical' proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a 'complete' understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 17 proteins with N-terminus FNR domain and C-terminus 2Fe-2S domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicate their plausible roles in degradation of aromatic moieties. PMID:24616557

  3. Analysis of multi-domain hypothetical proteins containing iron-sulphur clusters and fad ligands reveal rieske dioxygenase activity suggesting their plausible roles in bioremediation

    PubMed Central

    Sathyanarayanan, Nitish; Nagendra, Holenarasipur Gundurao

    2012-01-01

    ‘Conserved hypothetical’ proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a ‘complete’ understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 40 proteins with N-terminus 2Fe-2S domain and C-terminus FNR domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicating their plausible roles in degradation of aromatic moieties. PMID:23275712

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

    SciTech Connect

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M.

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiOx domains are titrated on TiOx–SiO2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiOx, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H2O2 activation occurs. We use this method to determine the active site densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h-1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.

  5. Mechanistic and bioinformatic investigation of a conserved active site helix in α-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.

    PubMed

    Casey, Ashley K; Hicks, Michael A; Johnson, Jordyn L; Babbitt, Patricia C; Frantom, Patrick A

    2014-05-13

    The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

  6. 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

  7. Evolution of anatase surface active sites probed by in situ sum-frequency phonon spectroscopy

    PubMed Central

    Cao, Yue; Chen, Shiyou; Li, Yadong; Gao, Yi; Yang, Deheng; Shen, Yuen Ron; Liu, Wei-Tao

    2016-01-01

    Surface active sites of crystals often govern their relevant surface chemistry, yet to monitor them in situ in real atmosphere remains a challenge. Using surface-specific sum-frequency spectroscopy, we identified the surface phonon mode associated with the active sites of undercoordinated titanium ions and conjoint oxygen vacancies, and used it to monitor them on anatase (TiO2) (101) under ambient conditions. In conjunction with theory, we determined related surface structure around the active sites and tracked the evolution of oxygen vacancies under ultraviolet irradiation. We further found that unlike in vacuum, the surface oxygen vacancies, which dominate the surface reactivity, are strongly regulated by ambient gas molecules, including methanol and water, as well as weakly associated species, such as nitrogen and hydrogen. The result revealed a rich interplay between prevailing ambient species and surface reactivity, which can be omnipresent in environmental and catalytic applications of titanium dioxides. PMID:27704049

  8. 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.

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

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  10. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

    PubMed

    Cannon, J L; Labno, C M; Bosco, G; Seth, A; McGavin, M H; Siminovitch, K A; Rosen, M K; Burkhardt, J K

    2001-08-01

    Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site. PMID:11520460

  11. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

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

    SciTech Connect

    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/mg 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.

  13. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

    PubMed

    Dramsi, Shaynoor; Scheid, Michael P; Maiti, Arpita; Hojabrpour, Payman; Chen, Xianming; Schubert, Kathryn; Goodlett, David R; Aebersold, Ruedi; Duronio, Vincent

    2002-02-22

    Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad. PMID:11717309

  14. Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

    SciTech Connect

    Fitzner, R.E.; Weiss, S.G.; Stegen, J.A.

    1994-06-01

    The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

  15. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

  16. 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

  17. 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. PMID:22492397

  18. The Life of Suggestions

    ERIC Educational Resources Information Center

    Pearce, Cathie

    2010-01-01

    Using the notion of a suggestion, or rather charting the life of suggestions, this article considers the happenings of chance and embodiment as the "problems that got away." The life of suggestions helps us to ask how connectivities are made, how desire functions, and how "immanence" rather than "transcendence" can open up the politics and ethics…

  19. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    SciTech Connect

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  20. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response.

  1. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  2. Structural Analysis of an Open Active Site Conformation of Nonheme Iron Halogenase CytC3

    SciTech Connect

    Wong, Cintyu; Galonic Fujimori, Danica; Walsh, Christopher T.; Drennan, Catherine L.

    2009-04-28

    CytC3, a member of the recently discovered class of nonheme Fe(II) and {alpha}-ketoglutarate ({alpha}KG)-dependent halogenases, catalyzes the double chlorination of l-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, {gamma},{gamma}-dichloroaminobutyrate. Unlike the majority of the Fe(II)-{alpha}KG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without {alpha}KG/Fe(II) have been solved to 2.2 {angstrom} resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound FeCl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

  3. Structure of the E. Coli Bifunctional GlmU Acetyltransferase Active Site with Substrates and Products

    SciTech Connect

    Olsen,L.; Vetting, M.; Roderick, S.

    2007-01-01

    The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO{sub 4} to form GlcNAc-1-PO{sub 4} and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO{sub 4}, and desulpho-CoA/GlcNAc-1-PO{sub 4}. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO{sub 4} is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

  4. Satellite imagery time series for the detection of looting activities at archaeological sites

    NASA Astrophysics Data System (ADS)

    Coluzzi, Rosa; Lasaponara, Rosa; Masini, Nicola

    2010-05-01

    Clandestine excavations is one of the biggest man-made risks which affect the archaeological heritage, especially in some countries of Southern America, Asia and Middle East. To contrast and limit this phenomenon a systematic monitoring is required. The protection of archaeological heritage from clandestine excavations is generally based on a direct surveillance, but it is time consuming and expensive for remote archaeological sites and non practicable in several countries due to military or political restrictions. In such conditions, Very high resolution (VHR) satellite imagery offer a suitable chance thanks to their global coverage and frequent revisitation times. This paper is focused on the results we obtained from ongoing research focused on the use of VHR satellite images for the identification and monitoring of looting. A time series of satellite images (QuickBird-2 and World-View-1) has been exploited to analyze and monitor archaeological looting in the Nasca Ceremonial Centre of Cahuachi (Peru) dating back between the 4th centurt B.C. and the 4th century A.D. The Cahuachi study case herein presented put in evidence the limits of VHR satellite imagery in detecting features linked to looting activity. This suggested to experience local spatial autocorrelation statistics which allowed us to improve the reliability of satellite in mapping looted area.

  5. 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.

  6. Analysis of Porcine Transcriptional Response to Salmonella enterica serovar Choleraesuis suggests novel targets of NFkappaB are activated in the Mesenteric Lymph Node

    PubMed Central

    Wang, Yanfang; Couture, Oliver P; Qu, Long; Uthe, Jolita J; Bearson, Shawn MD; Kuhar, Daniel; Lunney, Joan K; Nettleton, Dan; Dekkers, Jack CM; Tuggle, Christopher K

    2008-01-01

    Background Specific knowledge of the molecular pathways controlling host-pathogen interactions can increase our understanding of immune response biology as well as provide targets for drug development and genetic improvement of disease resistance. Toward this end, we have characterized the porcine transcriptional response to Salmonella enterica serovar Choleraesuis (S. Choleraesuis), a Salmonella serovar that predominately colonizes swine, yet can cause serious infections in human patients. Affymetrix technology was used to screen for differentially expressed genes in pig mesenteric lymph nodes (MLN) responding to infection with S. Choleraesuis at acute (8 hours (h), 24 h and 48 h post-inoculation (pi)) and chronic stages (21 days (d) pi). Results Analysis of variance with false discovery rate control identified 1,853 genes with significant changes in expression level (p-value < 0.01, q-value < 0.26, and fold change (FC) > 2) during infection as compared to un-inoculated control pigs. Down-regulation of translation-related genes at 8 hpi and 24 hpi implied that S. Choleraesuis repressed host protein translation. Genes involved in the Th1, innate immune/inflammation response and apoptosis pathways were induced significantly. However, antigen presentation/dendritic cell (DC) function pathways were not affected significantly during infection. A strong NFκB-dependent response was observed, as 58 known NFκB target genes were induced at 8, 24 and/or 48 hpi. Quantitative-PCR analyses confirmed the microarray data for 21 of 22 genes tested. Based on expression patterns, these target genes can be classified as an "Early" group (induced at either 8 or 24 hpi) and a "Late" group (induced only at 48 hpi). Cytokine activity or chemokine activity were enriched within the Early group genes GO annotations, while the Late group was predominantly composed of signal transduction and cell metabolism annotated genes. Regulatory motif analysis of the human orthologous promoters for

  7. Recurring Swarms of Deep Long Period Earthquakes in the Denali Volcanic Gap Suggest a Continuation of Volcanic Processes in the Absence of Active Volcanism

    NASA Astrophysics Data System (ADS)

    Holtkamp, S. G.; Ruppert, N. A.; Silwal, V.; Christensen, D. H.; Nye, C. J.

    2014-12-01

    Seismicity in the northern segment of the Denali Volcanic Gap clusters bimodally with depth, with dense clusters of earthquakes occurring in the subducting slab at >100 km depth beneath Denali, and within the crust north of the Denali fault at <20 km depth. On January 22, 2014, the Alaska Earthquake Center recorded a Deep Long Period earthquake (DLP), magnitude 1.7, at 40 km depth north of the Denali Fault. The epicenter for this event was <5 km of broadband station TRF, so the depth is well constrained. The DLP event is almost devoid of energy above 5 Hz. Receiver functions for stations TRF and SBL, both <10 km of the epicenter, show Moho depths of 36-40 km.We used waveforms of this DLP as a template event for network matched filtering, which identifies similar signals in continuous time series. We processed this template event from June 1999 to July 2014. We use several matches produced by this template as additional templates, iterating the process. Using this methodology, we identify over 300 DLP's. Events typically come in swarms lasting hours to days with no events exceeding magnitude 2. Swarms are separated by months to years of little detectable activity. A swarm of events on June 30, 2001 coincides with the Broadband Experiment Across the Alaska Range (BEAAR) seismic deployment, and was recorded by 15 broadband seismometers within 100 km of the epicenter. A preliminary waveform inversion for the focal mechanism of this event results in isotropic (implosive) and double couple components.We argue that these DLP's are evidence of magmatic or volatile movement through the sub-arc mantle wedge, even though there is no active volcanism at the surface. Relative relocations, utilizing cross correlated p- and s- waveforms, highlight a nest of seismicity with no structures such as planes or conduits. Lack of planar features, as well as the isotopic component and lack of strike slip to the focal mechanism, may argue against a deep extension of the Hines Creek or

  8. 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

  9. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

  10. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    NASA Astrophysics Data System (ADS)

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S.; Drakou, Evangelia G.; Pantis, John D.

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  11. Inhaled nitric oxide alters the distribution of blood flow in the healthy human lung, suggesting active hypoxic pulmonary vasoconstriction in normoxia

    PubMed Central

    Asadi, Amran K.; Sá, Rui Carlos; Kim, Nick H.; Theilmann, Rebecca J.; Hopkins, Susan R.; Buxton, Richard B.

    2014-01-01

    Hypoxic pulmonary vasoconstriction (HPV) is thought to actively regulate ventilation-perfusion (V̇a/Q̇) matching, reducing perfusion in regions of alveolar hypoxia. We assessed the extent of HPV in the healthy human lung using inhaled nitric oxide (iNO) under inspired oxygen fractions (FiO2) of 0.125, 0.21, and 0.30 (a hyperoxic stimulus designed to abolish HPV without the development of atelectasis). Dynamic measures of blood flow were made in a single sagittal slice of the right lung of five healthy male subjects using an arterial spin labeling (ASL) MRI sequence, following a block stimulus pattern (3 × 60 breaths) with 40 ppm iNO administered in the central block. The overall spatial heterogeneity, spatiotemporal variability, and regional pattern of pulmonary blood flow was quantified as a function of condition (FiO2 × iNO state). While spatial heterogeneity did not change significantly with iNO administration or FiO2, there were statistically significant increases in Global Fluctuation Dispersion, (a marker of spatiotemporal flow variability) when iNO was administered during hypoxia (5.4 percentage point increase, P = 0.003). iNO had an effect on regional blood flow that was FiO2 dependent (P = 0.02), with regional changes in the pattern of blood flow occurring in hypoxia (P = 0.007) and normoxia (P = 0.008) tending to increase flow to dependent lung at the expense of nondependent lung. These findings indicate that inhaled nitric oxide significantly alters the distribution of blood flow in both hypoxic and normoxic healthy subjects, and suggests that some baseline HPV may indeed be present in the normoxic lung. PMID:25429099

  12. Characterization of a DNA binding activity in DNAse I hypersensitive site 4 of the human globin locus control region.

    PubMed Central

    Walters, M; Kim, C; Gelinas, R

    1991-01-01

    A portion of the beta-globin Locus Control Region (LCR), which included DNAse I hypersensitive site 4 (HS4), was analyzed for its interactions with nuclear extracts and its contribution to LCR activity in a functional assay. In gel retardation assays, a short fragment from HS4 formed complexes with nuclear extracts from both erythroid and nonerythroid cells, and a core protected sequence 5'GACTGGC3' was revealed by DNAse I protection and methylation interference studies. This sequence resembles the binding sites of CCAAT-family members. Purified CP-2 but not CP-1 was shown to bind this HS4 sequence in a gel shift reaction, suggesting that the HS4 binding activity shares some sequence specificity with the CCAAT-factor family. Utilizing a transient expression assay in murine erythroleukemia cells, steady-state RNA levels were measured from pairs of LCR constructs linked to distinguishable beta-globin reporter genes. A short DNA fragment from HS4 which included the binding site for this novel binding activity accounted for most of the contribution to high level expression made by the entire HS4 region. Images PMID:1923823

  13. Automated docking of {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides in the glucoamylase active site

    SciTech Connect

    Countinho, P.M.; Reilly, P.J.; Dowd, M.K.

    1998-06-01

    Low-energy conformers of five {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides were flexibly docked into the glucoamylase active site using AutoDock 2.2. To ensure that all significant conformational space was searched, the starting trisaccharide conformers for docking were all possible combinations of the corresponding disaccharide low-energy conformers. All docked trisaccharides occupied subsites {minus}1 and +1 in very similar modes to those of corresponding nonreducing-end disaccharides. For linear substrates, full binding at subsite +2 occurred only when the substrate reducing end was {alpha}-(1,4)-linked, with hydrogen-bonding with the hydroxy-methyl group being the only polar interaction there. Given the absence of other important interactions at this subsite, multiple substrate conformations are allowed. For the one docked branched substrate, steric hindrance in the {alpha}-(1,6)-glycosidic oxygen suggests that the active-site residues have to change position for hydrolysis to occur. Subsite +1 of the glucoamylase active site allows flexibility in binding but, at least in Aspergillus glucoamylases, subsite +2 selectively binds substrates {alpha}-(1,4)-linked between subsites +1 and +2. Enzyme engineering to limit substrate flexibility at subsite +2 could improve glucoamylase industrial properties.

  14. Activity of radon (222Rn) in the lower atmospheric surface layer of a typical rural site in south India

    NASA Astrophysics Data System (ADS)

    Kumar, K. Charan; Prasad, T. Rajendra; Ratnam, M. Venkat; Nagaraja, Kamsali

    2016-09-01

    Analysis of one year measurements of in situ radon (222Rn) and its progenies along with surface air temperature, relative humidity and pressure near to the Earth's surface has been carried out for the first time at the National Atmospheric Research Laboratory (NARL, 13.5∘N and 79.2∘E) located in a rural site in Gadanki, south India. The dataset was analysed to understand the behaviour of radon in relation to the surface air temperature and relative humidity at a rural site. It was observed that over a period of the 24 hours in a day, the activity of radon and its progenies reaches a peak in the morning hours followed by a remarkable decrease in the afternoon hours. Relatively, a higher concentration of radon was observed at NARL during fair weather days, and this can be attributed to the presence of rocky hills and dense vegetation surrounding the site. The high negative correlation between surface air temperature and activity of radon (R = - 0.70, on an annual scale) suggests that dynamical removal of radon due to increased vertical mixing is one of the most important controlling processes of the radon accumulation in the atmospheric surface layer. The annual averaged activity of radon was found to be 12.01±0.66 Bq m-3 and 4.25±0.18 Bq m-3 for its progenies, in the study period.

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

    PubMed Central

    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. PMID:27276217

  16. 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. PMID:27276217

  17. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

  18. Analysis of active site residues of the antiviral protein from summer leaves from Phytolacca americana by site-directed mutagenesis.

    PubMed

    Poyet, J L; Hoeveler, A; Jongeneel, C V

    1998-12-30

    The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinant E. coli. On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active site mutants. PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced in E. coli and assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate. All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs). We conclude that none of the proposed mechanisms is entirely consistent with the data presented here.

  19. Pi-interaction tuning of the active site properties of metalloproteins.

    PubMed

    Yanagisawa, Sachiko; Crowley, Peter B; Firbank, Susan J; Lawler, Anne T; Hunter, David M; McFarlane, William; Li, Chan; Kohzuma, Takamitsu; Banfield, Mark J; Dennison, Christopher

    2008-11-19

    The influence of pi-interactions with a His ligand have been investigated in a family of copper-containing redox metalloproteins. The Met16Phe and Met16Trp pseudoazurin, and Leu12Phe spinach and Leu14Phe Phormidium laminosum plastocyanin variants possess active-site pi-contacts between the introduced residue and His81 and His87/92 respectively. The striking overlap of the side chain of Phe16 in the Met16Phe variant and that of Met16 in wild type pseudoazurin identifies that this position provides an important second coordination sphere interaction in both cases. His-ligand protonation and dissociation from Cu(I) occurs in the wild type proteins resulting in diminished redox activity, providing a [H(+)]-driven switch for regulating electron transfer. The introduced pi-interaction has opposing effects on the pKa for the His ligand in pseudoazurin and plastocyanin due to subtle differences in the pi-contact, stabilizing the coordinated form of pseudoazurin whereas in plastocyanin protonation and dissociation is favored. Replacement of Pro36, a residue that has been suggested to facilitate structural changes upon His ligand protonation, with a Gly, has little effect on the pKa of His87 in spinach plastocyanin. The mutations at Met16 have a significant influence on the reduction potential of pseudoazurin. Electron self-exchange is enhanced, whereas association with the physiological partner, nitrite reductase, is only affected by the Met16Phe mutation, but kcat is halved in both the Met16Phe and Met16Trp variants. Protonation of the His ligand is the feature most affected by the introduction of a pi-interaction.

  20. Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2013-01-01

    The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with 2 Mg2+ ions and cis-2-fluorogeranyl diphosphate at 1.85 Å and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally-occurring, non-canonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only 2 Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor. PMID:23844678

  1. The controlled relay of multiple protons required at the active site of nitrogenase.

    PubMed

    Dance, Ian

    2012-07-01

    The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

  2. Size Dependence of Atomically Precise Gold Nanoclusters in Chemoselective Hydrogenation and Active Site Structure

    SciTech Connect

    Li, Gao; Jiang, Deen; Kumar, Santosh; Chen, Yuxiang; Jin, Rongchao

    2014-01-01

    We here investigate the catalytic properties of water-soluble Aun(SG)m nanocluster catalysts (H-SG = glutathione) of different sizes, including Au15(SG)13, Au18(SG)14, Au25(SG)18, Au38(SG)24, and captopril-capped Au25(Capt)18 nanoclusters. These Aun(SR)m nanoclusters (-SR represents thiolate generally) are used as homogeneous catalysts (i.e., without supports) in the chemoselective hydrogenation of 4-nitrobenzaldehyde (4-NO2PhCHO) to 4-nitrobenzyl alcohol (4-NO2PhCH2OH) in water with H2 gas (20 bar) as the hydrogen source. These nanocluster catalysts, except Au18(SG)14, remain intact after the catalytic reaction, evidenced by UV-vis spectra which are characteristic of each sized nanoclusters and thus serve as spectroscopic fingerprints . We observe a drastic size-dependence and steric effect of protecting ligands on the gold nanocluster catalysts in the hydrogenation reaction. Density functional theory (DFT) modeling of the 4-nitrobenzaldehyde adsorption shows that both the CHO and NO2 groups are in close interact with the S-Au-S staples on the gold nanocluster surface; the adsorption of the 4-nitrobenzaldehyde molecule on the four different sized Aun(SR)m nanoclusters are moderately strong and similar in strength. The DFT results suggest that the catalytic activity of the Aun(SR)m nanoclusters is primarily determined by the surface area of the Au nanocluster, consistent with the observed trend of the conversion of 4-nitrobenzaldehyde versus the cluster size. Overall, this work offers the molecular insight into the hydrogenation of 4-nitrobenzaldehyde and the catalytically active site structure on gold nanocluster catalysts.

  3. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  4. Immobilized low-activity waste site borehole 299-E17-21

    SciTech Connect

    Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

    1998-08-01

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area.

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

    PubMed Central

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

    2016-01-01

    CphA is a Zn2+-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. PMID:27616327

  6. 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

    2016-01-01

    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. PMID:27616327

  7. 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.

  8. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  9. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    Cytochrome P450 monooxygenases are useful biocatalysts for C-H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

  10. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    Cytochrome P450 monooxygenases are useful biocatalysts for C-H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  11. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    PubMed

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

  12. CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways.

    PubMed

    Wang, Yang-Gang; Cantu, David C; Lee, Mal-Soon; Li, Jun; Glezakou, Vassiliki-Alexandra; Rousseau, Roger

    2016-08-24

    We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations. PMID:27480512

  13. Methyl Acetate Synthesis by Esterification on the Modified Ferrierite: Correlation of Acid Sites Measured by Pyridine IR and NH3-TPD for Steady-State Activity.

    PubMed

    Park, Jae Hyun; Pang, Changhyun; Chung, Chan-Hwa; Bae, Jong Wook

    2016-05-01

    The amounts of Brønsted acid sites on K, P, and Zr-modified microporous Ferrierite zeolite were investigated through pyridine FT-IR and NH3-TPD analyses. P-modified Ferrierite showed a superior catalytic activity for methyl acetate synthesis by esterification of methanol and acetic acid. The catalytic activity at steady-state with the acidic properties of as-prepared catalysts was well correlated with the results of pyridine FT-IR (intensity ratio of Brønsted acid sites to total acid sites) compared with that of NH3-TPD. The results can suggest the proper and simple method to estimate the esterification activity at steady-state using the measured acid sites on the as-prepared zeolites. PMID:27483801

  14. Immunogold evidence suggests that endoplasmic reticulum is the site of protamine-type protein synthesis and participates in translocation of these proteins into the nucleus during Chara vulgaris spermiogenesis.

    PubMed

    Popłonska, Katarzyna; Kwiatkowska, Maria; Wojtczak, Agnieszka; Polit, Justyna

    2009-03-01

    During spermiogenesis of an alga Chara vulgaris, which in many aspects resembles that of animals, histones are replaced by protamine-type proteins. Our earlier immunocytochemical studies showed that this replacement started during the short stage V of spermiogenesis, when electronograms revealed an extensive system of cisternae and vesicles of endoplasmic reticulum (ER). The present studies revealed at stage V intensive incorporation of labeled (3)H-arginine and (3)H-lysine quickly translocating into a nucleus visualized with pulse-chase autoradiography of semithin sections. The immunogold technique with the use of the antibodies to protamine-type proteins isolated from Chara tomentosa show that both ER cisternae and vesicles are labeled with gold grains, which are absent from the spermatids not treated with the antibodies; thus, the ER is probably the site of the protamine-type protein synthesis. These proteins then are translocated to a nucleus through ER channels connected with the nuclear envelope, as suggested by gold labeling of an inner membrane of the nuclear envelope adjacent to condensed chromatin. The above results correspond with those of other authors showing that in animals, protamines bind with lamin B receptors localized in the inner membrane of the nuclear envelope. A hypothesis has been put forward that during Chara spermiogenesis the inner membrane of the nuclear envelope invaginates into a nucleus together with protamine-type proteins, which become separated from the membrane and penetrate into chromatin.

  15. 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. PMID:21542861

  16. Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

    PubMed Central

    Teigelkamp, S; Newman, A J; Beggs, J D

    1995-01-01

    Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP. Images PMID:7781612

  17. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

  18. Active sites and mechanisms for direct oxidation of benzene to phenol over carbon catalysts.

    PubMed

    Wen, Guodong; Wu, Shuchang; Li, Bo; Dai, Chunli; Su, Dang Sheng

    2015-03-23

    The direct oxidation of benzene to phenol with H2 O2 as the oxidizer, which is regarded as an environmentally friendly process, can be efficiently catalyzed by carbon catalysts. However, the detailed roles of carbon catalysts, especially what is the active site, are still a topic of debate controversy. Herein, we present a fundamental consideration of possible mechanisms for this oxidation reaction by using small molecular model catalysts, Raman spectra, static secondary ion mass spectroscopy (SIMS), DFT calculations, quasi in situ ATR-IR and UV spectra. Our study indicates that the defects, being favorable for the formation of active oxygen species, are the active sites for this oxidation reaction. Furthermore, one type of active defect, namely the armchair configuration defect was successfully identified.

  19. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    SciTech Connect

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  20. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    PubMed Central

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M.; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease. PMID:24667638

  1. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  2. Coulombic effects of remote subsites on the active site of ribonuclease A.

    PubMed

    Fisher, B M; Schultz, L W; Raines, R T

    1998-12-15

    The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is lined with cationic residues that interact with a bound nucleic acid. Those residues interacting with the phosphoryl groups comprise the P0, P1, and P2 subsites, with the scissile P-O5' bond residing in the P1 subsite. Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the substrate were characterized previously [Fisher, B. M., Ha, J.-H., and Raines, R. T. (1998) Biochemistry 37, 12121-12132]. Here, the interactions between these subsites and the active-site residues His12 and His119 are described in detail. A protein variant in which the cationic residues in these subsites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were replaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP). Structures of K7A/R10A/K66A RNase A and the K7A/R10A/K66A RNase A.3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 A, respectively. There is little observable change between these structures and that of wild-type RNase A, either free or with bound 3'-cytidine monophosphate. K7A/R10A/K66A RNase A was evaluated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites. In comparison to the wild-type enzyme, the value of kcat was decreased by 5-fold and that of kcat/Km was decreased 10-fold, suggesting that these remote subsites interact with the active site. These interactions were characterized by determining the pKa values of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant. The side chains of Lys7, Arg10, and Lys66 depress the pKa values of these histidine residues, and this depression is sensitive to the salt concentration. In addition, the P0 and P2 subsites influence the interaction of His12 and His119 with each other, as demonstrated by changes in the cooperativity that gives rise to microscopic

  3. 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

  4. Chemistry Curricula. Course Suggestions.

    ERIC Educational Resources Information Center

    American Chemical Society, Washington, DC.

    Listings of suggested topics aimed at helping university and college faculties plan courses in the main areas of the chemistry curricula are provided. The suggestions were originally offered as appendices to the American Chemical Society's (ACS) Committee on Professional Training's 1983 guidelines for ACS-approved schools. The course data included…

  5. 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

    1993-08-01

    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.

  6. Evidence for surface Ag + complexes as the SERS-active sites on Ag electrodes

    NASA Astrophysics Data System (ADS)

    Watanabe, T.; Kawanami, O.; Honda, K.; Pettinger, B.

    1983-12-01

    Evidence is given that SERS-active sites at Ag electrodes are associated with Ag + ions, forming sparingly soluble surface complexes with ligands such as pyridine molecules and halide ions. Such surface Ag + complexes contribute a factor of >800 to the overall (10 7-fold) enhancement, possibly via a resonance Raman effect.

  7. Strategies and Activities for Using Local Communities as Environmental Education Sites.

    ERIC Educational Resources Information Center

    Roth, Charles E.; Lockwood, Linda G.

    Presented are over 100 environmental education activities which use the local community for a learning site and resource. These lessons are grouped under seven topical headings: (1) biological neighbors, (2) physical environs, (3) built environs, (4) social environs, (5) understanding ourselves, (6) influencing change, and (7) improvement and…

  8. 78 FR 18576 - Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-27

    ... Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection Instrument... information collection requirements and provide the requested data in the desired format. ED is soliciting... collection instrument will be used to collect specific information/performance data for analysis of...

  9. Penicillin Use in Meningococcal Disease Management: Active Bacterial Core Surveillance Sites, 2009

    PubMed Central

    Blain, Amy E.; Mandal, Sema; Wu, Henry; MacNeil, Jessica R.; Harrison, Lee H.; Farley, Monica M.; Lynfield, Ruth; Miller, Lisa; Nichols, Megin; Petit, Sue; Reingold, Arthur; Schaffner, William; Thomas, Ann; Zansky, Shelley M.; Anderson, Raydel; Harcourt, Brian H.; Mayer, Leonard W.; Clark, Thomas A.; Cohn, Amanda C.

    2016-01-01

    In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines. PMID:27704009

  10. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    ERIC Educational Resources Information Center

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  11. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the.... BOEM may issue one or more commercial wind energy leases in the WEA. The competitive lease process...

  12. 77 FR 3460 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to carry... for reimbursement. DOE amended the final rule on June 3, 2003, (68 FR 32955) to adopt several... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department...

  13. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

    ... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to carry... for reimbursement. DOE amended the final rule on June 3, 2003, (68 FR 32955) to adopt several... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department...

  14. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  15. Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity.

    PubMed

    Azevedo, Michelle de C S; Rosa, Patricia S; Soares, Cleverson T; Fachin, Luciana R V; Baptista, Ida Maria F D; Woods, William J; Garlet, Gustavo P; Trombone, Ana Paula F; Belone, Andrea de F F

    2015-01-01

    Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis. PMID:26700881

  16. Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

    PubMed Central

    Azevedo, Michelle de C. S.; Rosa, Patricia S.; Soares, Cleverson T.; Fachin, Luciana R. V.; Baptista, Ida Maria F. D.; Woods, William J.; Garlet, Gustavo P.

    2015-01-01

    Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis. PMID:26700881

  17. Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity.

    PubMed

    Azevedo, Michelle de C S; Rosa, Patricia S; Soares, Cleverson T; Fachin, Luciana R V; Baptista, Ida Maria F D; Woods, William J; Garlet, Gustavo P; Trombone, Ana Paula F; Belone, Andrea de F F

    2015-01-01

    Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.

  18. Spectroscopic studies of single and double variants of M ferritin: lack of conversion of a biferrous substrate site into a cofactor site for O2 activation.

    PubMed

    Kwak, Yeonju; Schwartz, Jennifer K; Haldar, Suranjana; Behera, Rabindra K; Tosha, Takehiko; Theil, Elizabeth C; Solomon, Edward I

    2014-01-28

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes.

  19. 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 abi