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Sample records for active site functional

  1. 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.

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

    PubMed Central

    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. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

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

    PubMed

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

    2014-10-01

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

  4. Synergistic effect between defect sites and functional groups on the hydrolysis of cellulose over activated carbon.

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

    The chemical oxidation of activated carbon by H2 O2 and H2 SO4 is investigated, structural and chemical modifications are characterized, and the materials are used as catalysts for the hydrolysis of cellulose. Treatment with H2 O2 enlarges the pore size and imparts functional groups such as phenols, lactones, and carboxylic acids. H2 SO4 treatment targets the edges of carbon sheets primarily, and this effect is more pronounced with a higher temperature. Adsorption isotherms demonstrate that the adsorption of oligomers on functionalized carbon is dominated by van der Waals forces. The materials treated chemically are active for the hydrolysis of cellulose despite the relative weakness of most of their acid sites. It is proposed that a synergistic effect between defect sites and functional groups enhances the activity by inducing a conformational change in the glucan chains if they are adsorbed at defect sites. This activates the glycosidic bonds for hydrolysis by in-plane functional groups.

  5. Comparison of protein active site structures for functional annotation of proteins and drug design.

    PubMed

    Powers, Robert; Copeland, Jennifer C; Germer, Katherine; Mercier, Kelly A; Ramanathan, Viswanathan; Revesz, Peter

    2006-10-01

    Rapid and accurate functional assignment of novel proteins is increasing in importance, given the completion of numerous genome sequencing projects and the vastly expanding list of unannotated proteins. Traditionally, global primary-sequence and structure comparisons have been used to determine putative function. These approaches, however, do not emphasize similarities in active site configurations that are fundamental to a protein's activity and highly conserved relative to the global and more variable structural features. The Comparison of Protein Active Site Structures (CPASS) database and software enable the comparison of experimentally identified ligand-binding sites to infer biological function and aid in drug discovery. The CPASS database comprises the ligand-defined active sites identified in the protein data bank, where the CPASS program compares these ligand-defined active sites to determine sequence and structural similarity without maintaining sequence connectivity. CPASS will compare any set of ligand-defined protein active sites, irrespective of the identity of the bound ligand. Proteins 2006. (c) 2006 Wiley-Liss, Inc.

  6. Functional biomimetic models for the active site in the respiratory enzyme cytochrome c oxidase.

    PubMed

    Collman, James P; Decréau, Richard A

    2008-11-07

    A functional analog of the active site in the respiratory enzyme, cytochrome c oxidase (CcO) reproduces every feature in CcO's active site: a myoglobin-like heme (heme a3), a distal tridentate imidazole copper complex (Cu(B)), a phenol (Tyr244), and a proximal imidazole. When covalently attached to a liquid-crystalline SAM film on an Au electrode, this functional model continuously catalyzes the selective four-electron reduction of dioxygen at physiological potential and pH, under rate-limiting electron flux (as occurs in CcO).

  7. The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions

    PubMed Central

    Pires, Virginia M. R; Flint, James E; Pinheiro, Benedita A; Goyal, Arun; Prates, José A. M; Izumi, Atsushi; Stålbrand, Henrik; Morland, Carl; Cartmell, Alan; Kolenova, Katarina; Topakas, Evangelos; Dodson, Eleanor J; Bolam, David N; Davies, Gideon J; Fontes, Carlos M. G. A; Gilbert, Harry J

    2009-01-01

    Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing

  8. Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

    DOE PAGES

    Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

    2014-12-09

    Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the

  9. Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

    SciTech Connect

    Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; Lutz, Stefan

    2014-12-09

    Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the contribution of

  10. DNA Sequence Constraints Define Functionally Active Steroid Nuclear Receptor Binding Sites in Chromatin.

    PubMed

    Coons, Laurel A; Hewitt, Sylvia C; Burkholder, Adam B; McDonnell, Donald P; Korach, Kenneth S

    2017-10-01

    Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin-interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications. Copyright © 2017 Endocrine Society.

  11. Protein function annotation with Structurally Aligned Local Sites of Activity (SALSAs).

    PubMed

    Wang, Zhouxi; Yin, Pengcheng; Lee, Joslynn S; Parasuram, Ramya; Somarowthu, Srinivas; Ondrechen, Mary Jo

    2013-01-01

    The prediction of biochemical function from the 3D structure of a protein has proved to be much more difficult than was originally foreseen. A reliable method to test the likelihood of putative annotations and to predict function from structure would add tremendous value to structural genomics data. We report on a new method, Structurally Aligned Local Sites of Activity (SALSA), for the prediction of biochemical function based on a local structural match at the predicted catalytic or binding site. Implementation of the SALSA method is described. For the structural genomics protein PY01515 (PDB ID 2aqw) from Plasmodium yoelii, it is shown that the putative annotation, Orotidine 5'-monophosphate decarboxylase (OMPDC), is most likely correct. SALSA analysis of YP_001304206.1 (PDB ID 3h3l), a putative sugar hydrolase from Parabacteroides distasonis, shows that its active site does not bear close resemblance to any previously characterized member of its superfamily, the Concanavalin A-like lectins/glucanases. It is noted that three residues in the active site of the thermophilic beta-1,4-xylanase from Nonomuraea flexuosa (PDB ID 1m4w), Y78, E87, and E176, overlap with POOL-predicted residues of similar type, Y168, D153, and E232, in YP_001304206.1. The substrate recognition regions of the two proteins are rather different, suggesting that YP_001304206.1 is a new functional type within the superfamily. A structural genomics protein from Mycobacterium avium (PDB ID 3q1t) has been reported to be an enoyl-CoA hydratase (ECH), but SALSA analysis shows a poor match between the predicted residues for the SG protein and those of known ECHs. A better local structural match is obtained with Anabaena beta-diketone hydrolase (ABDH), a known β-diketone hydrolase from Cyanobacterium anabaena (PDB ID 2j5s). This suggests that the reported ECH function of the SG protein is incorrect and that it is more likely a β-diketone hydrolase. A local site match provides a more compelling

  12. Protein function annotation with Structurally Aligned Local Sites of Activity (SALSAs)

    PubMed Central

    2013-01-01

    Background The prediction of biochemical function from the 3D structure of a protein has proved to be much more difficult than was originally foreseen. A reliable method to test the likelihood of putative annotations and to predict function from structure would add tremendous value to structural genomics data. We report on a new method, Structurally Aligned Local Sites of Activity (SALSA), for the prediction of biochemical function based on a local structural match at the predicted catalytic or binding site. Results Implementation of the SALSA method is described. For the structural genomics protein PY01515 (PDB ID 2aqw) from Plasmodium yoelii, it is shown that the putative annotation, Orotidine 5'-monophosphate decarboxylase (OMPDC), is most likely correct. SALSA analysis of YP_001304206.1 (PDB ID 3h3l), a putative sugar hydrolase from Parabacteroides distasonis, shows that its active site does not bear close resemblance to any previously characterized member of its superfamily, the Concanavalin A-like lectins/glucanases. It is noted that three residues in the active site of the thermophilic beta-1,4-xylanase from Nonomuraea flexuosa (PDB ID 1m4w), Y78, E87, and E176, overlap with POOL-predicted residues of similar type, Y168, D153, and E232, in YP_001304206.1. The substrate recognition regions of the two proteins are rather different, suggesting that YP_001304206.1 is a new functional type within the superfamily. A structural genomics protein from Mycobacterium avium (PDB ID 3q1t) has been reported to be an enoyl-CoA hydratase (ECH), but SALSA analysis shows a poor match between the predicted residues for the SG protein and those of known ECHs. A better local structural match is obtained with Anabaena beta-diketone hydrolase (ABDH), a known β-diketone hydrolase from Cyanobacterium anabaena (PDB ID 2j5s). This suggests that the reported ECH function of the SG protein is incorrect and that it is more likely a β-diketone hydrolase. Conclusions A local site match

  13. Site-specific phosphorylation induces functionally active conformation in the intrinsically disordered N-terminal activation function (AF1) domain of the glucocorticoid receptor.

    PubMed

    Garza, Anna M S; Khan, Shagufta H; Kumar, Raj

    2010-01-01

    Intrinsically disordered (ID) regions are disproportionately higher in cell signaling proteins and are predicted to have much larger frequency of phosphorylation sites than ordered regions, suggesting an important role in their regulatory capacity. In this study, we show that AF1, an ID activation domain of the glucocorticoid receptor (GR), adopts a functionally folded conformation due to its site-specific phosphorylation by p38 mitogen-activated protein kinase, which is involved in apoptotic and gene-inductive events initiated by the GR. Further, we show that site-specific phosphorylation-induced secondary and tertiary structure formation specifically facilitates AF1's interaction with critical coregulatory proteins and subsequently its transcriptional activity. These data demonstrate a mechanism through which ID activation domain of the steroid receptors and other similar transcription factors may adopt a functionally active conformation under physiological conditions.

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

    PubMed

    Hanson, Brett; Westin, Charles; Rosa, Mario; Grier, Alexander; Osipovitch, Mikhail; MacDonald, Madolyn L; Dodge, Greg; Boli, Paule M; Corwin, Cyprian W; Kessler, Haeja; McKay, Talia; Bernstein, Herbert J; Craig, Paul A

    2014-03-27

    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. 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. 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.

  15. Molybdenum and tungsten oxygen transferases--and functional diversity within a common active site motif.

    PubMed

    Pushie, M Jake; Cotelesage, Julien J; George, Graham N

    2014-01-01

    Molybdenum and tungsten are the only second and third-row transition elements with a known function in living organisms. The molybdenum and tungsten enzymes show common structural features, with the metal being bound by a pyranopterin-dithiolene cofactor called molybdopterin. They catalyze a variety of oxygen transferase reactions coupled with two-electron redox chemistry in which the metal cycles between the +6 and +4 oxidation states usually with water, either product or substrate, providing the oxygen. The functional roles filled by the molybdenum and tungsten enzymes are diverse; for example, they play essential roles in microbial respiration, in the uptake of nitrogen in green plants, and in human health. Together, the enzymes form a superfamily which is among the most prevalent known, being found in all kingdoms of life. This review discusses what is known of the active site structures and the mechanisms, together with some recent insights into the evolution of these important enzyme systems.

  16. Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

    PubMed Central

    Kaltenbach, Miriam; Jackson, Colin J; Campbell, Eleanor C; Hollfelder, Florian; Tokuriki, Nobuhiko

    2015-01-01

    Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks. DOI: http://dx.doi.org/10.7554/eLife.06492.001 PMID:26274563

  17. Site-specific Acetylation of the Proteasome Activator REGγ Directs Its Heptameric Structure and Functions*

    PubMed Central

    Liu, Jiang; Wang, Ying; Li, Lei; Zhou, Li; Wei, Haibin; Zhou, Qingxia; Liu, Jian; Wang, Weicang; Ji, Lei; Shan, Peipei; Wang, Yan; Yang, Yuanyuan; Jung, Sung Yun; Zhang, Pei; Wang, Chuangui; Long, Weiwen; Zhang, Bianhong; Li, Xiaotao

    2013-01-01

    The proteasome activator REGγ has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner. A recent comparative analysis of REGγ expression in mouse and human tissues reveals a unique pattern of REGγ in specific cell types, suggesting undisclosed functions and biological importance of this molecule. Despite the emerging progress made in REGγ-related studies, how REGγ function is regulated remains to be explored. In this study, we report for the first time that REGγ can be acetylated mostly on its lysine 195 (Lys-195) residue by CREB binding protein (CBP), which can be reversed by sirtuin 1 (SIRT1) in mammalian cells. Site-directed mutagenesis abrogated acetylation at Lys-195 and significantly attenuated the capability of REGγ to degrade its target substrates, p21 and hepatitis C virus core protein. Mechanistically, acetylation at Lys-195 is important for the interactions between REGγ monomers and ultimately influences REGγ heptamerization. Biological analysis of cells containing REGγ-WT or REGγ-K195R mutant indicates an impact of acetylation on REGγ-mediated regulation of cell proliferation and cell cycle progression. These findings reveal a previously unknown mechanism in the regulation of REGγ assembly and activity, suggesting a potential venue for the intervention of the ubiquitin-independent REGγ proteasome activity. PMID:23612972

  18. Prevalence of syn nucleobases in the active sites of functional RNAs

    PubMed Central

    Sokoloski, Joshua E.; Godfrey, Stephanie A.; Dombrowski, Sarah E.; Bevilacqua, Philip C.

    2011-01-01

    Biological RNAs, like their DNA counterparts, contain helical stretches, which have standard Watson-Crick base pairs in the anti conformation. Most functional RNAs also adopt geometries with far greater complexity such as bulges, loops, and multihelical junctions. Occasionally, nucleobases in these regions populate the syn conformation wherein the base resides close to or over the ribose sugar, which leads to a more compact state. The importance of the syn conformation to RNA function is largely unknown. In this study, we analyze 51 RNAs with tertiary structure, including aptamers, riboswitches, ribozymes, and ribosomal RNAs, for number, location, and properties of syn nucleobases. These RNAs represent the set of nonoverlapping, moderate- to high-resolution structures available at present. We find that syn nucleobases are much more common among purines than pyrimidines, and that they favor C2′-endo-like conformations especially among those nucleobases in the intermediate syn conformation. Strikingly, most syn nucleobases participate in tertiary stacking and base-pairing interactions: Inspection of RNA structures revealed that the majority of the syn nucleobases are in regions assigned to function, with many syn nucleobases interacting directly with a ligand or ribozyme active site. These observations suggest that judicious placement of conformationally restricted nucleotides biased into the syn conformation could enhance RNA folding and catalysis. Such changes could also be useful for locking RNAs into functionally competent folds for use in X-ray crystallography and NMR. PMID:21873463

  19. Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

    PubMed

    Turbeville, Tracy D; Zhang, Junshun; Adams, W Christopher; Hunter, Gregory A; Ferreira, Gloria C

    2011-07-01

    5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Exploring functionally related enzymes using radially distributed properties of active sites around the reacting points of bound ligands

    PubMed Central

    2012-01-01

    Background Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance) of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities. Results We showed that radial distribution functions (RDFs), which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments. Conclusions Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites. PMID:22536854

  1. Noncovalent intermolecular interactions between dehydroepiandrosterone and the active site of human dehydroepiandrosterone sulphotransferase: A density functional theory based treatment

    NASA Astrophysics Data System (ADS)

    Astani, Elahe; Heshmati, Emran; Chen, Chun-Jung; Hadipour, Nasser L.; Shekarsaraei, Setareh

    2016-04-01

    A theoretical study was performed to characterize noncovalent intermolecular interactions, especially hydrogen bond (HB), in the active site of enzyme human dehydroepiandrosterone sulphotransferase (SULT2A1/DHEA) using the local (M06-L) and hybrid (M06, M06-2X) meta-GGA functionals of density functional theory (DFT). Results revealed that DHEA is able to form HBs with residues His99, Tyr231, Met137 and Met16 in the active site of the SULT2A1/DHEA. It was found that DHEA interacts with the other residues through electrostatic and Van der Waals interactions.

  2. Functional evidence of distinct ATP activation sites at the human P2X7 receptor

    PubMed Central

    Klapperstück, Manuela; Büttner, Cora; Schmalzing, Günther; Markwardt, Fritz

    2001-01-01

    The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X7 receptor (hP2X7), an N-terminally hexahistidyl-tagged hP2X7 receptor (His-hP2X7), or a truncated His-hP2X7 receptor (His-hP2X7ΔC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique. The activation time course of the wild-type hP2X7 receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component. The amplitude of the exponentially activating current component of the wild-type hP2X7 receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 μm free ATP4-, respectively. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 μm. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X7 or His-hP2X7ΔC. Our data indicate that the hP2X7 receptor possesses at least two types of activation sites, which differ in ATP4- sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites. PMID:11432989

  3. Dissection of Pol II trigger loop function and Pol II activity-dependent control of start site selection in vivo.

    PubMed

    Kaplan, Craig D; Jin, Huiyan; Zhang, Ivan Liang; Belyanin, Andrey

    2012-01-01

    Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process.

  4. Zinc cysteine active sites of metalloproteins: A density functional theory and x-ray absorption fine structure study

    NASA Astrophysics Data System (ADS)

    Dimakis, Nicholas; Farooqi, Mohammed Junaid; Garza, Emily Sofia; Bunker, Grant

    2008-03-01

    Density functional theory (DFT) and x-ray absorption fine structure (XAFS) spectroscopy are complementary tools for the biophysical study of active sites in metalloproteins. DFT is used to compute XAFS multiple scattering Debye Waller factors, which are then employed in genetic algorithm-based fitting process to obtain a global fit to the XAFS in the space of fitting parameters. Zn-Cys sites, which serve important functions as transcriptional switches in Zn finger proteins and matrix metalloproteinases, previously have proven intractable by this method; here these limitations are removed. In this work we evaluate optimal DFT nonlocal functionals and basis sets for determining optimal geometries and vibrational densities of states of mixed ligation Zn(His)4-n(Cys)n sites. Theoretical results are compared to experimental XAFS measurements and Raman spectra from the literature and tabulated for use.

  5. Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo in Saccharomyces cerevisiae

    PubMed Central

    Dolinski, Kara; Scholz, Christian; Muir, R. Scott; Rospert, Sabine; Schmid, Franz X.; Cardenas, Maria E.; Heitman, Joseph

    1997-01-01

    Cyclophilin and FK506 binding protein (FKBP) accelerate cis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions. PMID:9362068

  6. Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

    PubMed Central

    Chen, J W; Evans, B R; Yang, S H; Araki, H; Oshima, Y; Jayaram, M

    1992-01-01

    The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant. Images PMID:1508181

  7. Discarding Functional Residues from the Substitution Table Improves Predictions of Active Sites within Three-Dimensional Structures

    PubMed Central

    Gong, Sungsam; Blundell, Tom L.

    2008-01-01

    Substitutions of individual amino acids in proteins may be under very different evolutionary restraints depending on their structural and functional roles. The Environment Specific Substitution Table (ESST) describes the pattern of substitutions in terms of amino acid location within elements of secondary structure, solvent accessibility, and the existence of hydrogen bonds between side chains and neighbouring amino acid residues. Clearly amino acids that have very different local environments in their functional state compared to those in the protein analysed will give rise to inconsistencies in the calculation of amino acid substitution tables. Here, we describe how the calculation of ESSTs can be improved by discarding the functional residues from the calculation of substitution tables. Four categories of functions are examined in this study: protein–protein interactions, protein–nucleic acid interactions, protein–ligand interactions, and catalytic activity of enzymes. Their contributions to residue conservation are measured and investigated. We test our new ESSTs using the program CRESCENDO, designed to predict functional residues by exploiting knowledge of amino acid substitutions, and compare the benchmark results with proteins whose functions have been defined experimentally. The new methodology increases the Z-score by 98% at the active site residues and finds 16% more active sites compared with the old ESST. We also find that discarding amino acids responsible for protein–protein interactions helps in the prediction of those residues although they are not as conserved as the residues of active sites. Our methodology can make the substitution tables better reflect and describe the substitution patterns of amino acids that are under structural restraints only. PMID:18833291

  8. AutoDock-GIST: Incorporating Thermodynamics of Active-Site Water into Scoring Function for Accurate Protein-Ligand Docking.

    PubMed

    Uehara, Shota; Tanaka, Shigenori

    2016-11-23

    Water plays a significant role in the binding process between protein and ligand. However, the thermodynamics of water molecules are often underestimated, or even ignored, in protein-ligand docking. Usually, the free energies of active-site water molecules are substantially different from those of waters in the bulk region. The binding of a ligand to a protein causes a displacement of these waters from an active site to bulk, and this displacement process substantially contributes to the free energy change of protein-ligand binding. The free energy of active-site water molecules can be calculated by grid inhomogeneous solvation theory (GIST), using molecular dynamics (MD) and the trajectory of a target protein and water molecules. Here, we show a case study of the combination of GIST and a docking program and discuss the effectiveness of the displacing gain of unfavorable water in protein-ligand docking. We combined the GIST-based desolvation function with the scoring function of AutoDock4, which is called AutoDock-GIST. The proposed scoring function was assessed employing 51 ligands of coagulation factor Xa (FXa), and results showed that both scoring accuracy and docking success rate were improved. We also evaluated virtual screening performance of AutoDock-GIST using FXa ligands in the directory of useful decoys-enhanced (DUD-E), thus finding that the displacing gain of unfavorable water is effective for a successful docking campaign.

  9. Elucidating the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts: A density functional theory study

    NASA Astrophysics Data System (ADS)

    Wang, Ziyun; Liu, Xinyi; Rooney, D. W.; Hu, P.

    2015-10-01

    The dehydrogenation of cyclohexanol to cyclohexanone is very important in the manufacture of nylon. Copper-based catalysts are the most popular catalysts for this reaction, and on these catalysts the reaction mechanism and active site are in debate. In order to elucidate the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts, density functional theory with dispersion corrections were performed on up to six facets of copper in two different oxidation states: monovalent copper and metallic copper. By calculating the surface energies of these facets, Cu(111) and Cu2O(111) were found to be the most stable facets for metallic copper and for monovalent copper, respectively. On these two facets, all the possible elementary steps in the dehydrogenation pathway of cyclohexanol were calculated, including the adsorption, dehydrogenation, hydrogen coupling and desorption. Two different reaction pathways for dehydrogenation were considered on both surfaces. It was revealed that the dehydrogenation mechanisms are different on these two surfaces: on Cu(111) the hydrogen belonging to the hydroxyl is removed first, then the hydrogen belonging to the carbon is subtracted, while on Cu2O(111) the hydrogen belonging to the carbon is removed followed by the subtraction of the hydrogen in the hydroxyl group. Furthermore, by comparing the energy profiles of these two surfaces, Cu2O(111) was found to be more active for cyclohexanol dehydrogenation than Cu(111). In addition, we found that the coordinatively unsaturated copper sites on Cu2O(111) are the reaction sites for all the steps. Therefore, the coordinatively unsaturated copper site on Cu2O(111) is likely to be the active site for cyclohexanol dehydrogenation on the copper-based catalysts.

  10. Crystal Structures and Functional Characterization of Wild Type and Active Sites Mutants of CYP101D1

    PubMed Central

    Batabyal, Dipanwita; Poulos, Thomas L.

    2014-01-01

    Although CYP101D1 and P450cam catayze the same reaction at a similar rate and share strikingly similar active site architectures, there are significance functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what can differ yet maintain high catalytic efficiency. Crystal structures of the cyanide complex of wild type CYP101D1 and it active site mutants, D259N and T260A, have been solved. The conformational changes in CYP101D1 upon cyanide binding are very similar to P450cam indicating a similar mechanism for proton delivery during oxygen activation using solvent assisted proton transfer. The D259N-CN− complex shows a perturbed solvent structure compared to wild type which is similar to what was observed in the oxy-complex of the corresonding D251N mutant in P450cam. As in P450cam the T260A mutant is highly uncoupled while the D259N gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provides a strucutral basis for understanding these functional differences. PMID:24261604

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

    PubMed Central

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

    2010-01-01

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

  12. Elicitor Activity of a Fungal Endopolygalacturonase in Tobacco Requires a Functional Catalytic Site and Cell Wall Localization

    PubMed Central

    Boudart, Georges; Charpentier, Myriam; Lafitte, Claude; Martinez, Yves; Jauneau, Alain; Gaulin, Elodie; Esquerré-Tugayé, Marie-Thérèse; Dumas, Bernard

    2003-01-01

    CLPG1, an endopolygalacturonase (endoPG) gene of Colletotrichum lindemuthianum, was transferred to tobacco (Nicotiana tabacum) leaves by using the Agrobacterium tumefaciens transient delivery system. The following four constructs were prepared: CLPG1, with or without its signal peptide (SP; PG1, PG1ΔSP); CLPG1 with the tobacco expansin1 SP instead of its own SP (Exp::PG1ΔSP); and a mutated version of the latter on two amino acids potentially involved in the catalytic site of CLPG1 (D202N/D203N). Chlorotic and necrotic lesions appeared 5 to 7 d postinfiltration, exclusively in response to CLPG1 fused to the expansin SP. The lesions were correlated to the production of an active enzyme. Necrosis-inducing activity, as well as endoPG activity, were completely abolished by site-directed mutagenesis. Ultrastructural immunocytolocalization experiments indicated that the expansin SP addressed CLPG1 to the cell wall. Staining of parenchyma cells revealed the progressive degradation of pectic material in junction zones and middle lamella as a function of time after infiltration, ultimately leading to cell separation. A 30% decrease in the GalUA content of the cell walls was simultaneously recorded, thereby confirming the hydrolytic effect of CLPG1 on pectic polysaccharides, in planta. The elicitor activity of CLPG1 was further illustrated by the induction of defense responses comprising active oxygen species and β-1,3-glucanase activity, before leaf necrosis. Altogether, the data demonstrate that an appropriate SP and a functional catalytic site are required for the proper expression and elicitor activity of the fungal endoPG CLPG1 in tobacco. PMID:12529518

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

    SciTech Connect

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

    2012-04-18

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

  14. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.

    PubMed

    Gangopadhyay, Soumyashree A; Losito, Erica L; Hougland, James L

    2014-01-21

    Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.

  15. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

    The List 9 displays the sequence of activities undertaken at active CERCLIS sites. An active site is one at which site assessment, removal, remedial, enforcement, cost recovery, or oversight activities are being planned or conducted.

  16. Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

    PubMed

    Qureshi, Shabir H; Yang, Likui; Manithody, Chandrashekhara; Bae, Jong-Sup; Rezaie, Alireza R

    2008-09-01

    Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.

  17. Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification

    PubMed Central

    West, Nicholas P.; Chow, Frances M. E.; Randall, Elizabeth J.; Wu, Jing; Chen, Jian; Ribeiro, Jose M. C.; Britton, Warwick J.

    2009-01-01

    Discovery and characterization of novel secreted enzymes of Mycobacterium tuberculosis are important for understanding the pathogenesis of one of the most important human bacterial pathogens. The proteome of M. tuberculosis contains over 400 potentially secreted proteins, the majority of which are uncharacterized. A family of seven cutinase-like proteins (CULPs) was identified by bioinformatic analysis, expressed and purified from Escherichia coli, and characterized in terms of their enzymatic activities. These studies revealed a functional diversity of enzyme classes based on differential preferences for substrate chain length. One member, Culp1, exhibited strong esterase activity, 40-fold higher than that of Culp6, which had strong activity as a lipase. Another, Culp4, performed moderately as an esterase and weakly as a lipase. Culp6 lipase activity was optimal above pH 7.0, and fully maintained to pH 8.5. None of the CULP members exhibited cutinase activity. Site-directed mutagenesis of each residue of the putative catalytic triad in Culp6 confirmed that each was essential for activity toward all fatty acid chain lengths of nitrophenyl esters and lipolytic function. Culp1 and Culp2 were present only in culture supernatants of M. tuberculosis, while Culp6, which is putatively essential for mycobacterial growth, was retained in the cell wall, suggesting the proteins play distinct roles in mycobacterial biology.—West, N. P., Chow, F. M. E., Randall, E. J., Wu, J., Chen, J., Ribeiro, J. M. C., Britton, W. J. Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification. PMID:19225166

  18. Monoclonal anti-idiotypic antibodies as functional internal images of enzyme active sites: production of a catalytic antibody with a cholinesterase activity.

    PubMed Central

    Izadyar, L; Friboulet, A; Remy, M H; Roseto, A; Thomas, D

    1993-01-01

    Monoclonal antibody 9A8 was selected by immunizing mice with AE-2, a monoclonal antibody directed against the active site of acetylcholinesterase. In accordance with the idiotypic network theory, monoclonal anti-idiotypic antibody 9A8 displayed internal-image properties of the original immunogen, the acetylcholinesterase active site. Hydrolysis of acetylthiocholine and related esters of thiocholine by 9A8 follows saturation kinetics and kinetic parameters were determined. The hydrolytic activity is characterized by a lowered kcat value (81 s-1) and an increased Km value (0.6 mM) when compared with the original enzyme. However, the rate acceleration (kcat/kuncat = 4.15 x 10(8) remains higher than for the esterase activities usually described for catalytic antibodies directed against transition-state analogs. The 9A8 activity exhibits a relaxation of specificity toward both substrates and inhibitors. This specificity does not correspond to a known enzymatic activity. The anti-idiotypic approach should be valuable for producing different structural and functional copies of the same enzyme active site. This should allow further insights into structure-activity relationships. Furthermore, use of chemically modified enzymes as immunogens may result in anti-idiotypic antibodies with catalytic activities not found in the native enzymes. PMID:8415624

  19. SitesIdentify: a protein functional site prediction tool

    PubMed Central

    2009-01-01

    Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify), based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/ PMID:19922660

  20. Identification of the active site of DS-epimerase 1 and requirement of N-glycosylation for enzyme function.

    PubMed

    Pacheco, Benny; Maccarana, Marco; Goodlett, David R; Malmström, Anders; Malmström, Lars

    2009-01-16

    Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.

  1. Tuning Pt and Cu sites population inside functionalized UiO-67 MOF by controlling activation conditions.

    PubMed

    Braglia, L; Borfecchia, E; Lomachenko, K A; Bugaev, A L; Guda, A A; Soldatov, A V; Bleken, B T L; Øien-Ødegaard, S; Olsbye, U; Lillerud, K P; Bordiga, S; Agostini, G; Manzoli, M; Lamberti, C

    2017-09-08

    The exceptional thermal and chemical stability of the UiO-66, -67 and -68 classes of isostructural MOFs [J. Am. Chem. Soc., 2008, 130, 13850] makes them ideal materials for functionalization purposes aimed at introducing active centres for potential application in heterogeneous catalysis. We previously demonstrated that a small fraction (up to 10%) of the linkers in the UiO-67 MOF can be replaced by bipyridine-dicarboxylate (bpydc) moieties exhibiting metal-chelating ability and enabling the grafting of Pt(ii) and Pt(iv) ions in the MOF framework [Chem. Mater., 2015, 27, 1042] upon interaction with PtCl2 or PtCl4 precursors. Herein we extend this functionalization approach in two directions. First, we show that by controlling the activation of the UiO-67-Pt we can move from a material hosting isolated Pt(ii) sites anchored to the MOF framework with Pt(ii) exhibiting two coordination vacancies (potentially interesting for C-H bond activation) to the formation of very small Pt nanoparticles hosted inside the MOF cavities (potentially interesting for hydrogenation reactions). The second direction consists of the extension of the approach to the insertion of Cu(ii), obtained via interaction with CuCl2, and exhibiting interesting redox properties. All materials have been characterized by in situ X-ray absorption spectroscopy at the Pt L3- and Cu K-edges.

  2. The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.

    PubMed

    Martinez, Salette; Wu, Rui; Sanishvili, Ruslan; Liu, Dali; Holz, Richard

    2014-01-29

    Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp(2)) form to a tetrahedral form (sp(3)). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase-boronic acid complexes represent a "snapshot" of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys(113)-OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.

  3. Simultaneous presence of both open metal sites and free functional organic sites in a noncentrosymmetric dynamic metal-organic framework with bimodal catalytic and sensing activities.

    PubMed

    Saha, Rajat; Joarder, Biplab; Roy, Anupam Singha; Manirul Islam, Sk; Kumar, Sanjay

    2013-12-02

    Assimilation of open metal sites (OMSs) and free functional organic sites (FOSs) with a framework strut has opened up a new route for the fabrication of novel metal-organic materials, thereby providing a unique opportunity to explore their multiple functionalities. A new metal-organic framework (MOF), {[Cu(ina)2(H2O)][Cu(ina)2(bipy)]·2H2O}n (1) (ina=isonicotinate, bipy=4,4'-bipyridine), has been synthesized and characterized. Complex 1 is crystallized in the orthorhombic noncentrosymmetric space group Aba2 and consists of two different 2D coordination polymers, [Cu(ina)2(H2O)]n and [Cu(ina)2(bipy)]n, with entrapped solvent water molecules. Hydrogen-bonding interactions assemble these two different 2D coordination layers in a single-crystal structure with interdigitation of pendant 4,4'-bipy from one layer into the groove of another. Upon removal of guest molecules, 1 undergoes a structural transformation in single-crystal-to-single-crystal fashion with expansion of the effective void space. Each metal center is five-coordinated and thus can potentially behave as an OMS, and the free pyridyl groups of pendant 4,4'-bipy moieties and free -C=O groups can act as free FOSs. Thus, owing to presence of both OMSs and free FOSs, the framework exhibits multifunctional properties. Owing to the presence of OMSs, the framework can act as a Lewis acid catalyst as well as a small-molecule sensor material, and in a similar way, owing to the presence of free FOSs, it performs as a Lewis base catalyst and a cation sensor material. Furthermore, owing to noncentrosymmetry with large polarity along a particular direction, it shows strong second-harmonic generation/nonlinear optical (SHG-NLO) activity. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Density functional study on UV/VIS spectra of copper-protein active sites: the effect of mutations.

    PubMed

    Swart, Marcel; Johansson, Mikael P

    2012-09-01

    UV/VIS Electron excitation spectra have been computed for large, realistic model systems of the blue copper protein family. Fully quantum-chemical calculations at the density-functional theory level employing polarized triple-ζ basis sets have been performed on systems of over 120 atoms, without symmetry. Different mutants, with the ligating methionine of the wild type Cu center exchanged for histidine (M121H) and glutamine (M121Q), have been investigated in order to obtain insight about how the influence of the exact surrounding milieu of the Cu-atom affects the computed spectrum. With sufficiently large model sizes, inclusion of the environment by using continuum solvation models do not change the spectra significantly. More direct and rigorous treatments are needed to reliably assess the effect of the surrounding protein on the electronic structure of the active sites. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  5. Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY.

    PubMed

    Zhang, Huijun; Guo, Wenbin; Xu, Changan; Zhou, Hongbo; Chen, Xinhua

    2013-12-14

    Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.

  6. Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.

    PubMed

    Eiamphungporn, Warawan; Soonsanga, Sumarin; Lee, Jin-Won; Helmann, John D

    2009-03-01

    Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

  7. Linking structure to function: The search for active sites in non-platinum group metal oxygen reduction reaction catalysts

    SciTech Connect

    Holby, Edward F.; Zelenay, Piotr

    2016-05-17

    Atomic-scale structures of oxygen reduction reaction (ORR) active sites in non-platinum group metal (non-PGM) catalysts, made from pyrolysis of carbon, nitrogen, and transition-metal (TM) precursors have been the subject of continuing discussion in the fuel cell electrocatalysis research community. We found that quantum chemical modeling is a path forward for understanding of these materials and how they catalyze the ORR. Here, we demonstrate through literature examples of how such modeling can be used to better understand non-PGM ORR active site relative stability and activity and how such efforts can also aid in the interpretation of experimental signatures produced by these materials.

  8. Linking structure to function: The search for active sites in non-platinum group metal oxygen reduction reaction catalysts

    SciTech Connect

    Holby, Edward F.; Zelenay, Piotr

    2016-05-17

    Atomic-scale structures of oxygen reduction reaction (ORR) active sites in non-platinum group metal (non-PGM) catalysts, made from pyrolysis of carbon, nitrogen, and transition-metal (TM) precursors have been the subject of continuing discussion in the fuel cell electrocatalysis research community. We found that quantum chemical modeling is a path forward for understanding of these materials and how they catalyze the ORR. Here, we demonstrate through literature examples of how such modeling can be used to better understand non-PGM ORR active site relative stability and activity and how such efforts can also aid in the interpretation of experimental signatures produced by these materials.

  9. The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism.

    PubMed

    Dance, Ian

    2015-11-07

    Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O(+) from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 Å) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-HO hydrogen bonds, and the occurrence of long (up to 3.6 Å) separations between contiguous water molecules. Both steps involve low potential energy barriers, <7 kcal mol(-1). During operation of the Grotthuss mechanism in nitrogenase there are substantial displacements of water molecules along the chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The

  10. Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

    PubMed

    Álvarez, Lucía; Lewis-Ballester, Ariel; Roitberg, Adrián; Estrin, Darío A; Yeh, Syun-Ru; Marti, Marcelo A; Capece, Luciana

    2016-05-17

    Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

  11. Density functional theory study of model complexes for the revised nitrate reductase active site in Desulfovibrio desulfuricans NapA.

    PubMed

    Hofmann, Matthias

    2009-09-01

    [Mo(SSCH3)(S2C2(CH3)2)2](x) complexes with charges x between -3 and +3 were investigated by density functional theory computations as minimal nitrate reductase active-site models. The strongly reduced species (x = -2, -3) exist preferentially as pentacoordinate sulfo complexes separated from a thiolate anion. The oxidized extremes (x > 0) clearly prefer hexacoordinate complexes with an eta(2)-MeSS ligand. Among the neutral and especially for the singly negatively charged species structures with eta(2)-MeSS and eta(1)-MeSS ligands are energetically close to the sulfo methyl sulfide complex without SS bonding. For x = -1 the three isomers lie in a 1.5 kcal mol(-1) energy range. Putative mechanistic pathways for nitrate reduction from the literature were investigated computationally: (1) reduction at a pentacoordinate sulfo complex, (2) reduction at the ligand, and (3) reduction at the molybdenum center with an R-S-S ligand. All three pathways could be traced at least for some overall charges but no definite conclusion can be drawn about the mechanism. Complexes with larger dithiolato ligands were also computed in order to model the tricyclic metallopterin framework more accurately: the first heterocyclus (5,6-dihydro-2H-pyran) stabilizes the nitrate complex and the molybdenum oxo product complex by approximately 10 kcal mol(-1) and also reduces the activation barrier (by approximately 5 kcal mol(-1)). The effect of the second (1,2,3,4-tetrahydropyrazin) and third heterocyclus (2-amino-3H-pyrimidin-4-one) on the relative energies is relatively small. For bigger models derived from an experimental protein structure, nitrate reduction at a persulfo molybdenum(IV) complex fragment (mechanism 3) is clearly favored over the oxidation of a molybdenum-bound sulfur atom (mechanism 2). Mechanism 1 could not be investigated for the big models but seems the least favorable on the basis of the results from smaller models.

  12. KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

    PubMed

    Bihani, Subhash C; Chakravarty, Dhiman; Ballal, Anand

    2016-04-01

    Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

  14. Linking structure to function: The search for active sites in non-platinum group metal oxygen reduction reaction catalysts

    DOE PAGES

    Holby, Edward F.; Zelenay, Piotr

    2016-05-17

    Atomic-scale structures of oxygen reduction reaction (ORR) active sites in non-platinum group metal (non-PGM) catalysts, made from pyrolysis of carbon, nitrogen, and transition-metal (TM) precursors have been the subject of continuing discussion in the fuel cell electrocatalysis research community. We found that quantum chemical modeling is a path forward for understanding of these materials and how they catalyze the ORR. Here, we demonstrate through literature examples of how such modeling can be used to better understand non-PGM ORR active site relative stability and activity and how such efforts can also aid in the interpretation of experimental signatures produced by thesemore » materials.« less

  15. (Function of active-site residues of ribulosebisphosphate carboxylase/oxygenase, Stockholm, Sweden, and visit to Uppsala, Sweden, August 6--12, 1989): Foreign trip report

    SciTech Connect

    Hartman, F.C.

    1989-08-22

    The traveler participated in the 8th International Congress on Photosynthesis by presenting a paper entitled ''Function of Active-Site Residues of Ribulosebisphosphate Carboxylase/Oxygenase'' and by chairing a discussion session on the same enzyme. Presentation concerning biological CO/sub 2/ fixation, chemical modifications of proteins, 3D structure of proteins, and site-directed mutagenesis were relevant to ongoing investigations of the Protein Engineering Program at ORNL's Biology Division.

  16. Towards site-based protein functional annotations.

    PubMed

    Lei, Seak Fei; Huan, Jun

    2010-01-01

    The exact relationship between protein active centres and protein functions is unclear even after decades of intensive study. To improve functional prediction ability based on the local structures, we proposed three different methods. 1. We used Markov Random Field (MRF) to describe protein active region. 2. We developed filtering method that considers the local environment around the active sites. 3. We created multiple structure motifs by extending the motif to neighbouring residues. Our experiment results with enzyme families < 40% sequence identity demonstrated that our methods reduced random matches and could improve up to 70% of the functional annotation ability (using area under curve).

  17. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

    PubMed Central

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr215 in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr215, effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr215 can thus lead to a kinetic bottleneck in product release. PMID:27302031

  18. An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins.

    PubMed

    Harper, Angela F; Leuthaeuser, Janelle B; Babbitt, Patricia C; Morris, John H; Ferrin, Thomas E; Poole, Leslie B; Fetrow, Jacquelyn S

    2017-02-01

    Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially-MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method's novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated by

  19. An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins

    PubMed Central

    Babbitt, Patricia C.; Ferrin, Thomas E.

    2017-01-01

    Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially—MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method’s novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated

  20. Functional and mechanistic analyses of biomimetic aminoacyl transfer reactions in de novo designed coiled coil peptides via rational active site engineering.

    PubMed

    Leman, Luke J; Weinberger, Dana A; Huang, Zheng-Zheng; Wilcoxen, Keith M; Ghadiri, M Reza

    2007-03-14

    Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the epsilon-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to approximately 103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15N NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pK(a) depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes.

  1. An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

    PubMed Central

    Knutson, Stacy T.; Westwood, Brian M.; Leuthaeuser, Janelle B.; Turner, Brandon E.; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D.; Harper, Angela F.; Brown, Shoshana D.; Morris, John H.; Ferrin, Thomas E.; Babbitt, Patricia C.

    2017-01-01

    Abstract Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results. PMID:28054422

  2. An approach to functionally relevant clustering of the protein universe: Active site profile-based clustering of protein structures and sequences.

    PubMed

    Knutson, Stacy T; Westwood, Brian M; Leuthaeuser, Janelle B; Turner, Brandon E; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D; Harper, Angela F; Brown, Shoshana D; Morris, John H; Ferrin, Thomas E; Babbitt, Patricia C; Fetrow, Jacquelyn S

    2017-04-01

    Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification-amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two-Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure-Function Linkage Database, SFLD) self-identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self-identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well-curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP-identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F-measure and performance analysis on the enolase search results and comparison to GEMMA and SCI-PHY demonstrate that TuLIP avoids the over-division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results.

  3. Amplified Rate Acceleration by Simultaneous Up-Regulation of Multiple Active Sites in an Endo-Functionalized Porous Capsule.

    PubMed

    Kopilevich, Sivil; Müller, Achim; Weinstock, Ira A

    2015-10-14

    Using the hydrolysis of epoxides in water as a model reaction, the effect of multiple active sites on Michaelis-Menten compliant rate accelerations in a porous capsule is demonstrated. The capsule is a water-soluble Ih-symmetry Keplerate-type complex of the form, [{Mo(VI)6O21(H2O)6}12{Mo(V)2O4(L)}30](42-), in which 12 pentagonal "ligands," {(Mo(VI))Mo(VI)5O21(H2O)6}(6-), are coordinated to 30 dimolybdenum sites, {Mo(V)2O4L}(1+) (L = an endohedrally coordinated η(2)-bound carboxylate anion), resulting in 20 Mo9O9 pores. When "up-regulated" by removal of ca. one-third of the blocking ligands, L, an equal number of dimolybdenum sites are activated, and the newly freed-up space allows for encapsulation of nearly twice as many substrate guests, leading to a larger effective molarity (amplification), and an increase in the rate acceleration (k(cat)/k(uncat)) from 16,000 to an enzyme-like value of 182,800.

  4. A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

    PubMed

    Hatakeyama, Dai; Shoji, Masaki; Yamayoshi, Seiya; Hirota, Takenori; Nagae, Monami; Yanagisawa, Shin; Nakano, Masahiro; Ohmi, Naho; Noda, Takeshi; Kawaoka, Yoshihiro; Kuzuhara, Takashi

    2014-09-05

    The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Functional analyses for tRNase Z variants: an aspartate and a histidine in the active site are essential for the catalytic activity.

    PubMed

    Elbarbary, Reyad A; Takaku, Hiroaki; Nashimoto, Masayuki

    2008-12-01

    We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn(2+)-rescue analysis for Thermotoga maritima tRNase Z(S) has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z(S) variants and human tRNase Z(L) variants for cleavage activities on pre-tRNAs in the presence of Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.

  6. Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

    PubMed

    Ohmae, Eiji; Miyashita, Yurina; Tate, Shin-Ichi; Gekko, Kunihiko; Kitazawa, Soichiro; Kitahara, Ryo; Kuwajima, Kunihiro

    2013-12-01

    To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme. © 2013.

  7. Functional activation independently contributes to naming ability and relates to lesion site in post-stroke aphasia.

    PubMed

    Skipper-Kallal, Laura M; Lacey, Elizabeth H; Xing, Shihui; Turkeltaub, Peter E

    2017-04-01

    Language network reorganization in aphasia may depend on the degree of damage in critical language areas, making it difficult to determine how reorganization impacts performance. Prior studies on remapping of function in aphasia have not accounted for the location of the lesion relative to critical language areas. They rectified this problem by using a multimodal approach, combining multivariate lesion-symptom mapping and fMRI in chronic aphasia to understand the independent contributions to naming performance of the lesion and the activity in both hemispheres. Activity was examined during two stages of naming: covert retrieval, and overt articulation. Regions of interest were drawn based on over- and under-activation, and in areas where activity had a bivariate relationship with naming. Regressions then tested whether activation of these regions predicted naming ability, while controlling for lesion size and damage in critical left hemisphere naming areas, as determined by lesion-symptom mapping. Engagement of the right superior temporal sulcus (STS) and disengagement of the left dorsal pars opercularis (dPOp) during overt naming was associated with better than predicted naming performance. Lesions in the left STS prevented right STS engagement and resulted in persistent left dPOp activation. In summary, changes in activity during overt articulation independently relate to naming outcomes, controlling for stroke severity. Successful remapping relates to network disruptions that depend on the location of the lesion in the left hemisphere. Hum Brain Mapp 38:2051-2066, 2017. © 2017 Wiley Periodicals, Inc.

  8. Improvement in pump function with endocardial biventricular pacing increases with activation time at the left ventricular pacing site in failing canine hearts.

    PubMed

    Howard, Elliot J; Covell, James W; Mulligan, Lawrence J; McCulloch, Andrew D; Omens, Jeffrey H; Kerckhoffs, Roy C P

    2011-10-01

    Recently, attention has been focused on comparing left ventricular (LV) endocardial (ENDO) with epicardial (EPI) pacing for cardiac resynchronization therapy. However, the effects of ENDO and EPI lead placement at multiple sites have not been studied in failing hearts. We hypothesized that differences in the improvement of ventricular function due to ENDO vs. EPI pacing in dyssynchronous (DYSS) heart failure may depend on the position of the LV lead in relation to the original activation pattern. In six nonfailing and six failing dogs, electrical DYSS was created by atrioventricular sequential pacing of the right ventricular apex. ENDO was compared with EPI biventricular pacing at five LV sites. In failing hearts, increases in the maximum rate of LV pressure change (dP/dt; r = 0.64), ejection fraction (r = 0.49), and minimum dP/dt (r = 0.51), relative to DYSS, were positively correlated (P < 0.01) with activation time at the LV pacing site during ENDO but not EPI pacing. ENDO pacing at sites with longer activation delays led to greater improvements in hemodynamic parameters and was associated with an overall reduction in electrical DYSS compared with EPI pacing (P < 0.05). These findings were qualitatively similar for nonfailing hearts. Improvement in hemodynamic function increased with activation time at the LV pacing site during ENDO but not EPI pacing. At the anterolateral wall, end-systolic transmural function was greater with local ENDO compared with EPI pacing. ENDO pacing and intrinsic activation delay may have important implications for management of DYSS heart failure.

  9. Inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme.

    PubMed

    Knap, A K; Pratt, R F

    1991-01-01

    The pH-rate profile for inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate supports previous evidence [Knap & Pratt (1989) Proteins Struct. Funct. Genet. 6, 316-323] for the activation of the active-site thiol group by adjacent functional groups. The enhanced reactivity of iodoacetate, with respect to that of iodoacetamide, suggests the influence of a positive charge in the active site. The reactivity of iodoacetate is not affected by dissociation of an active-site functional group of pKa 6.7, which increases the reactivity of neutral reagents, probably because of a compensation phenomenon; it is, however, lost on dissociation of an acid of pKa 8.1. It is concluded that the active cysteine beta-lactamase has four functional groups at the active site, one nucleophilic thiolate of Cys-70, one neutral acid (most probably the carboxy group of Glu-166, from the crystal structures) and two cationic residues (most probably Lys-73 and Lys-234). A comparison of these results with the pH-dependence of reactivity of the native RTEM-2 beta-lactamase suggests that the active form of the latter enzyme is also monocationic, although the nucleophile (Ser-70) is likely to be neutral in this case and the carboxylic acid dissociated. A mechanism of class A beta-lactamase catalysis is discussed where the Glu-166 carboxylate acts as a general base/acid catalyst and Lys-73 is principally required for electrostatic stabilization of the anionic tetrahedral intermediate.

  10. New insights into the catalytic mechanism of histidine phosphatases revealed by a functionally essential arginine residue within the active site of the Sts phosphatases.

    PubMed

    San Luis, Boris; Nassar, Nicolas; Carpino, Nick

    2013-07-01

    Sts (suppressor of T-cell receptor signalling)-1 and Sts-2 are HPs (histidine phosphatases) that negatively regulate TCR (T-cell receptor) signalling pathways, including those involved in cytokine production. HPs play key roles in such varied biological processes as metabolism, development and intracellular signalling. They differ considerably in their primary sequence and substrate specificity, but possess a catalytic core formed by an invariant quartet of active-site residues. Two histidine and two arginine residues cluster together within the HP active site and are thought to participate in a two-step dephosphorylation reaction. To date there has been little insight into any additional residues that might play an important functional role. In the present study, we identify and characterize an additional residue within the Sts phosphatases (Sts-1 Arg383 or Sts-2 Arg369) that is critical for catalytic activity and intracellular function. Mutation of Sts-1 Arg383 to an alanine residue compromises the enzyme's activity and renders Sts-1 unable to suppress TCR-induced cytokine induction. Of the multiple amino acids substituted for Arg383, only lysine partially rescues the catalytic activity of Sts-1. Although Sts-1 Arg383 is conserved in all Sts homologues, it is only conserved in one of the two sub-branches of HPs. The results of the present study highlight an essential role for Sts-1 phosphatase activity in regulating T-cell activation and add a new dimension of complexity to our understanding of HP catalytic activity.

  11. Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

    PubMed Central

    Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

    2016-01-01

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  12. Invited award contribution for ACS Award in Inorganic Chemistry. Geometric and electronic structure contributions to function in bioinorganic chemistry: active sites in non-heme iron enzymes.

    PubMed

    Solomon, E I

    2001-07-16

    Spectroscopy has played a major role in the definition of structure/function correlations in bioinorganic chemistry. The importance of spectroscopy combined with electronic structure calculations is clearly demonstrated by the non-heme iron enzymes. Many members of this large class of enzymes activate dioxygen using a ferrous active site that has generally been difficult to study with most spectroscopic methods. A new spectroscopic methodology has been developed utilizing variable temperature, variable field magnetic circular dichroism, which enables one to obtain detailed insight into the geometric and electronic structure of the non-heme ferrous active site and probe its reaction mechanism on a molecular level. This spectroscopic methodology is presented and applied to a number of key mononuclear non-heme iron enzymes leading to a general mechanistic strategy for O2 activation. These studies are then extended to consider the new features present in the binuclear non-heme iron enzymes and applied to understand (1) the mechanism of the two electron/coupled proton transfer to dioxygen binding to a single iron center in hemerythrin and (2) structure/function correlations over the oxygen-activating enzymes stearoyl-ACP Delta9-desaturase, ribonucleotide reductase, and methane monooxygenase. Electronic structure/reactivity correlations for O2 activation by non-heme relative to heme iron enzymes will also be developed.

  13. Functional effects of diphosphomimetic mutations at cAbl-mediated phosphorylation sites on Rad51 recombinase activity.

    PubMed

    Alligand, Brendan; Le Breton, Magali; Marquis, Damien; Vallette, François; Fleury, Fabrice

    2017-08-01

    Homologous Recombination enables faithful repair of the deleterious double strand breaks of DNA. This pathway relies on Rad51 to catalyze homologous DNA strand exchange. Rad51 is known to be phosphorylated in a sequential manner on Y315 and then on Y54, but the effect of such phosphorylation on Rad51 function remains poorly understood. We have developed a phosphomimetic model in order to study all the phosphorylation states. With the purified phosphomimetic proteins we performed in vitro assays to determine the activity of Rad51. Here we demonstrate the inhibitory effect of the double phosphomimetic mutant and suggest that it may be due to a defect in nucleofilament formation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.

    PubMed

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

    2010-11-26

    Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.

  15. Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

    PubMed

    Thuresson, E D; Lakkides, K M; Rieke, C J; Sun, Y; Wingerd, B A; Micielli, R; Mulichak, A M; Malkowski, M G; Garavito, R M; Smith, W L

    2001-03-30

    Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually

  16. Calcium-manganese oxides as structural and functional models for active site in oxygen evolving complex in photosystem II: lessons from simple models.

    PubMed

    Najafpour, Mohammad Mahdi

    2011-01-01

    The oxygen evolving complex in photosystem II which induces the oxidation of water to dioxygen in plants, algae and certain bacteria contains a cluster of one calcium and four manganese ions. It serves as a model to split water by sunlight. Reports on the mechanism and structure of photosystem II provide a more detailed architecture of the oxygen evolving complex and the surrounding amino acids. One challenge in this field is the development of artificial model compounds to study oxygen evolution reaction outside the complicated environment of the enzyme. Calcium-manganese oxides as structural and functional models for the active site of photosystem II are explained and reviewed in this paper. Because of related structures of these calcium-manganese oxides and the catalytic centers of active site of the oxygen evolving complex of photosystem II, the study may help to understand more about mechanism of oxygen evolution by the oxygen evolving complex of photosystem II.

  17. Redox-active on-surface polymerization of single-site divalent cations from pure metals by a ketone-functionalized phenanthroline

    SciTech Connect

    Skomski, Daniel; Tempas, Christopher D.; Bukowski, Gregory S.; Smith, Kevin A.; Tait, Steven L.

    2015-03-14

    Metallic iron, chromium, or platinum mixing with a ketone-functionalized phenanthroline ligand on a single crystal gold surface demonstrates redox activity to a well-defined oxidation state and assembly into thermally stable, one dimensional, polymeric chains. The diverging ligand geometry incorporates redox-active sub-units and bi-dentate binding sites. The gold surface provides a stable adsorption environment and directs growth of the polymeric chains, but is inert with regard to the redox chemistry. These systems are characterized by scanning tunnelling microscopy, non-contact atomic force microscopy, and X-ray photoelectron spectroscopy under ultra-high vacuum conditions. The relative propensity of the metals to interact with the ketone group is examined, and it is found that Fe and Cr more readily complex the ligand than Pt. The formation and stabilization of well-defined transition metal single-sites at surfaces may open new routes to achieve higher selectivity in heterogeneous catalysts.

  18. Redox-active on-surface polymerization of single-site divalent cations from pure metals by a ketone-functionalized phenanthroline

    NASA Astrophysics Data System (ADS)

    Skomski, Daniel; Tempas, Christopher D.; Bukowski, Gregory S.; Smith, Kevin A.; Tait, Steven L.

    2015-03-01

    Metallic iron, chromium, or platinum mixing with a ketone-functionalized phenanthroline ligand on a single crystal gold surface demonstrates redox activity to a well-defined oxidation state and assembly into thermally stable, one dimensional, polymeric chains. The diverging ligand geometry incorporates redox-active sub-units and bi-dentate binding sites. The gold surface provides a stable adsorption environment and directs growth of the polymeric chains, but is inert with regard to the redox chemistry. These systems are characterized by scanning tunnelling microscopy, non-contact atomic force microscopy, and X-ray photoelectron spectroscopy under ultra-high vacuum conditions. The relative propensity of the metals to interact with the ketone group is examined, and it is found that Fe and Cr more readily complex the ligand than Pt. The formation and stabilization of well-defined transition metal single-sites at surfaces may open new routes to achieve higher selectivity in heterogeneous catalysts.

  19. Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

    PubMed

    Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

    2017-06-07

    Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

  20. Molecular and functional analysis of the XPBC/ERCC-3 promoter: transcription activity is dependent on the integrity of an Sp1-binding site.

    PubMed Central

    Ma, L; Weeda, G; Jochemsen, A G; Bootsma, D; Hoeijmakers, J H; van der Eb, A J

    1992-01-01

    The human XPBC/ERCC-3 gene, which corrects the excision-repair defect in xeroderma pigmentosum group B cells and the UV-sensitive CHO mutant 27-1 cells, appears to be expressed constitutively in various cell types and tissues. We have analysed the structure and functionality of the XPBC/ERCC-3 promoter. Transcription of the XPBC/ERCC-3 gene is initiated from heterogeneous sites, with a major startpoint mapped at position -54 (relative to the translation start codon ATG). The promoter region does not possess classical TATA and CAAT elements, but it is GC-rich and contains three putative Sp1-binding sites. In addition, there are two elements related to the cyclic AMP (cAMP)-response element (CRE) and the 12-O-tetradecanoyl phorbol-13-acetate-response element (TRE) in the 5'-flanking region. Transient expression analysis of XPBC/ERCC-3 promoter-CAT chimeric plasmids revealed that a 127-bp fragment, spanning position -129 to -3, is minimally required for the promoter activity. Transcription of the XPBC/ERCC-3 promoter depends on the integrity of a putative Sp1-binding site in close proximity to the major cap site. Band shift assays showed that this putative Sp1-binding site can interact specifically with a nuclear factor, most likely transcription factor Sp1 (or an Sp1-like factor) in vitro. Images PMID:1741247

  1. 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.

  2. Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1.

    PubMed

    Khan, Asad U; Rehman, M Tabish

    2015-11-02

    New Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned blaNDM-1 from an Enterobacter cloacae clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the Km was reduced, while the kcat was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔGD (o) and Tm of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.

    PubMed

    Itoh, Yuzuru; Bröcker, Markus J; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2014-04-17

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site.

    PubMed

    Hegeman, A D; Gross, J W; Frey, P A

    2001-06-05

    A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate-nucleotide but contains NAD(+) (PDB file ). From the model, two groups of active site residues were identified. The first group consists of Asp135(DEH), Glu136(DEH), Glu198(DEH), Lys199(DEH), and Tyr301(DEH). These residues are near the substrate-pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase. The second group of residues is Cys187(DEH), Asn190(DEH), and His232(DEH), which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases. The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis. In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed. Mutagenesis of each of these residues, with the exception of Cys187(DEH), which showed near-wild-type activity, clearly has important negative consequences for catalysis. The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism. Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions. To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose. Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation. The results are

  5. On the multiple functional roles of the active site histidine in catalysis and allosteric regulation of Escherichia coli glucosamine 6-phosphate deaminase.

    PubMed

    Montero-Morán, G M; Lara-González, S; Alvarez-Añorve, L I; Plumbridge, J A; Calcagno, M L

    2001-08-28

    side chain of Glu148 moves toward the active site. The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k(cat) versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting. This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range.

  6. Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing.

    PubMed

    Chen, Lie; Tian, Lijun; MacDonald, Stephen H-F; McClafferty, Heather; Hammond, Martin S L; Huibant, Jean-Marc; Ruth, Peter; Knaus, Hans-Guenther; Shipston, Michael J

    2005-09-30

    The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.

  7. The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase.

    PubMed

    Arjomand, Maryam Rezaei; Ahmadian, Gholamreza; Habibi-Rezaei, Mehran; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Moosavi-Movahedi, Ali Akbar; Amanlou, Massoud

    2017-05-01

    Here, we have studied the role of a histidine residue with the lowest solvent accessibility among other histidine residues at the end of a short connecting structure ((189)AELH(192)) of the catalytic domain of the exo-inulinase through creation of H192A mutant. Site-directed mutagenesis method was applied to create the mutant enzyme. Molecular dynamics (MD) simulations, spectroscopic, calorimetric and kinetics analysis were used to study the structural and functional consequences of His192 substitution. Accordingly, the thermo-stabilities and catalytic performance were decreased upon H192A mutation. In silico and experimental approaches evidently confirm that His192 residue of exo-inulinase possesses structural and functional importance regardless of the lack of direct interaction with the substrate or involvement in the catalytic activity of exo-inulinase. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Functional Mapping of the Lectin Activity Site on the β-Prism Domain of Vibrio cholerae Cytolysin

    PubMed Central

    Rai, Anand Kumar; Paul, Karan; Chattopadhyay, Kausik

    2013-01-01

    Vibrio cholerae cytolysin (VCC) is a prominent member in the family of β-barrel pore-forming toxins. It induces lysis of target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the β-Prism domain, we demonstrate that the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the β-Prism domain's lectin activity, implicated in the process of membrane pore formation by VCC. PMID:23209283

  9. Active site analysis of sortase A from Staphylococcus simulans indicates function in cleavage of putative cell wall proteins.

    PubMed

    Chen, Jian; Dong, Huihui; Murfin, Kristen E; Feng, Chunyan; Wu, Shaoqiang; Zheng, Beiwen

    2016-09-30

    Sortase mediated transpeptidation reactions play a significant role in covalent attachment of surface proteins to the cell wall of Gram-positive bacteria. Earlier studies have shown that sortase A (StrA) is required for the virulence of Staphylococci. The human pathogen Staphylococcus simulans CJ16 carries a putative sortase A (SsiStrA) encoding gene, but neither transpeptidation activity nor biochemical characteristics of SsiStrA have been investigated. Here, we identified and characterized StrA from coagulase-negative Staphylococci. SsiStrA was cloned and overexpressed in Escherichia coli BL21 in a soluble form. Size-exclusion chromatography, cross-linking and dynamic light scattering demonstrated that SsiStrA existed as monomer-dimer equilibrium in vitro. We further demonstrated that SsiStrA has sortase activity, and it recognized and cleaved the sorting motif LXPTG. H117, C180 and R193 residues were critical for enzyme activity, and calcium ions enhanced activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.

    PubMed Central

    Hunninghake, G W; Monks, B G; Geist, L J; Monick, M M; Monroy, M A; Stinski, M F; Webb, A C; Dayer, J M; Auron, P E; Fenton, M J

    1992-01-01

    Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation. Images PMID:1630455

  11. Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

    PubMed Central

    Lednicky, J; Folk, W R

    1992-01-01

    The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

  12. Organization and function of membrane contact sites.

    PubMed

    Helle, Sebastian C J; Kanfer, Gil; Kolar, Katja; Lang, Alexander; Michel, Agnès H; Kornmann, Benoît

    2013-11-01

    Membrane-bound organelles are a wonderful evolutionary acquisition of the eukaryotic cell, allowing the segregation of sometimes incompatible biochemical reactions into specific compartments with tailored microenvironments. On the flip side, these isolating membranes that crowd the interior of the cell, constitute a hindrance to the diffusion of metabolites and information to all corners of the cell. To ensure coordination of cellular activities, cells use a network of contact sites between the membranes of different organelles. These membrane contact sites (MCSs) are domains where two membranes come to close proximity, typically less than 30nm. Such contacts create microdomains that favor exchange between two organelles. MCSs are established and maintained in durable or transient states by tethering structures, which keep the two membranes in proximity, but fusion between the membranes does not take place. Since the endoplasmic reticulum (ER) is the most extensive cellular membrane network, it is thus not surprising to find the ER involved in most MCSs within the cell. The ER contacts diverse compartments such as mitochondria, lysosomes, lipid droplets, the Golgi apparatus, endosomes and the plasma membrane. In this review, we will focus on the common organizing principles underlying the many MCSs found between the ER and virtually all compartments of the cell, and on how the ER establishes a network of MCSs for the trafficking of vital metabolites and information. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Investigations and activities at superfund sites.

    PubMed

    Gochfeld, M; Burger, J

    1990-01-01

    Only a small proportion of the hazardous waste sites in the United States have been placed on the National Priority List (NPL), and these have become known as Superfund Sites. Many more sites exist on active industrial or military facilities, and a large number of sites remain to be discovered. It is useful to illustrate several hazardous waste sites to provide a clearer impression of the nature of the hazards, the kind of work done, and the proximity of residences to sites.

  14. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

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

  15. Chemical modification of the RTEM-1 thiol beta-lactamase by thiol-selective reagents: evidence for activation of the primary nucleophile of the beta-lactamase active site by adjacent functional groups.

    PubMed

    Knap, A K; Pratt, R F

    1989-01-01

    The RTEM-1 thiol beta-lactamase (Sigal, I.S., Harwood, B.G., Arentzen, R., Proc. Natl. Acad. Sci. U.S.A. 79:7157-7160, 1982) is inactivated by thiol-selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4'-dipyridyldisulfide, which modify the active site thiol group. The pH-rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The pKa of the active site thiol is therefore shown by the data to be below 4.0. This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol beta-lactamase is stabilized by two hydrogen-bond donors. One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys-73 ammonium group, while the identity of the other group, of pKa around 6.7, is less clear, but may be the conserved Glu-166 carboxylic acid. beta-Lactamase activity is associated with the EH2 form, and thus the beta-lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol beta-lactamase) and two acidic (hydrogen-bond donor) groups (one of which is likely to be the above-mentioned lysine ammonium group).

  16. Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

    PubMed

    Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-03-25

    Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology and designing enzymes with new functional capabilities. This article is protected by copyright. All rights reserved.

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

    DOE PAGES

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

    2016-05-23

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

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

    SciTech Connect

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

    2016-05-23

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Murphy, Jesse; Mullins, Elwood; Kappock, T.

    2016-05-01

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

  1. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1990-10-01

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

  2. 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.

  3. Formation and dimerization of the phosphodiesterase active site of the Pseudomonas aeruginosa MorA, a bi-functional c-di-GMP regulator.

    PubMed

    Phippen, Curtis William; Mikolajek, Halina; Schlaefli, Henry George; Keevil, Charles William; Webb, Jeremy Stephen; Tews, Ivo

    2014-12-20

    Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC-PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections. Copyright © 2014. Published by Elsevier B.V.

  4. Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase.

    PubMed

    Reschke, Stefan; Niks, Dimitri; Wilson, Heather; Sigfridsson, Kajsa G V; Haumann, Michael; Rajagopalan, K V; Hille, Russ; Leimkühler, Silke

    2013-11-19

    Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (kcat/KM) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt). X-ray absorption spectroscopy revealed a Mo(VI)-Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)O2Mo(VI)S2(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOMD4Ser was altered compared to those of SOMD4Ser and SOMD4Cys(wt), with g1 in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo-Se bond.

  5. A Casein Kinase II Phosphorylation Site in AtYY1 Affects Its Activity, Stability, and Function in the ABA Response

    PubMed Central

    Wu, Xiu-Yun; Li, Tian

    2017-01-01

    The phosphorylation and dephosphorylation of proteins are crucial in the regulation of protein activity and stability in various signaling pathways. In this study, we identified an ABA repressor, Arabidopsis Ying Yang 1 (AtYY1) as a potential target of casein kinase II (CKII). AtYY1 physically interacts with two regulatory subunits of CKII, CKB3, and CKB4. Moreover, AtYY1 can be phosphorylated by CKII in vitro, and the S284 site is the major CKII phosphorylation site. Further analyses indicated that S284 phosphorylation can enhance the transcriptional activity and protein stability of AtYY1 and hence strengthen the effect of AtYY1 as a negative regulator in the ABA response. Our study provides novel insights into the regulatory mechanism of AtYY1 mediated by CKII phosphorylation. PMID:28348572

  6. Requirements for functional models of the iron hydrogenase active site: D2/H2O exchange activity in ((mu-SMe)(mu-pdt)[Fe(CO)2(PMe3)]2+)[BF4-].

    PubMed

    Georgakaki, Irene P; Miller, Matthew L; Darensbourg, Marcetta Y

    2003-04-21

    Hydrogen uptake in hydrogenase enzymes can be assayed by H/D exchange reactivity in H(2)/D(2)O or H(2)/D(2)/H(2)O mixtures. Diiron(I) complexes that serve as structural models for the active site of iron hydrogenase are not active in such isotope scrambling but serve as precursors to Fe(II)Fe(II) complexes that are functional models of [Fe]H(2)ase. Using the same experimental protocol as used previously for ((mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-H(+) (Zhao et al. J. Am. Chem. Soc. 2001, 123, 9710), we now report the results of studies of ((mu-SMe)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-SMe(+), toward H/D exchange. The 1-SMe(+) complex can take up H(2) and catalyze the H/D exchange reaction in D(2)/H(2)O mixtures under photolytic, CO-loss conditions. Unlike 1-H(+), it does not catalyze H(2)/D(2) scrambling under anhydrous conditions. The molecular structure of 1-SMe(+) involves an elongated Fe.Fe separation, 3.11 A, relative to 2.58 A in 1-H(+). It is proposed that the strong SMe(-) bridging ligand results in catalytic activity localized on a single Fe(II) center, a scenario that is also a prominent possibility for the enzyme active site. The single requirement is an open site on Fe(II) available for binding of D(2) (or H(2)), followed by deprotonation by the external base H(2)O (or D(2)O).

  7. Structure-function analysis of the LytM domain of EnvC, an activator of cell wall remodeling at the Escherichia coli division site

    PubMed Central

    Peters, Nick T.; Morlot, Cécile; Yang, Desirée C.; Uehara, Tsuyoshi; Vernet, Thierry; Bernhardt, Thomas G.

    2013-01-01

    Proteins with LytM (Peptidase_M23) domains are broadly distributed in bacteria and have been implicated in a variety of important processes, including cell division and cell-shape determination. Most LytM-like proteins that have been structurally and/or biochemically characterized are metallo-endopeptidases that cleave crosslinks in the peptidoglycan (PG) cell wall matrix. Notable exceptions are the Escherichia coli cell division proteins EnvC and NlpD. These LytM factors are not hydrolases themselves, but instead serve as activators that stimulate PG cleavage by target enzymes called amidases to promote cell separation. Here we report the structure of the LytM domain from EnvC, the first structure of a LytM factor implicated in the regulation of PG hydrolysis. As expected, the fold is highly similar to that of other LytM proteins. However, consistent with its role as a regulator, the active site region is degenerate and lacks a catalytic metal ion. Importantly, genetic analysis indicates that residues in and around this degenerate active site are critical for amidase activation in vivo and in vitro. Thus, in the regulatory LytM factors, the apparent substrate binding pocket conserved in active metallo-endopeptidases has been adapted to control PG hydrolysis by another set of enzymes. PMID:23796240

  8. Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate.

    PubMed Central

    Meng, M.; Chane, T. L.; Sun, Y. J.; Hsiao, C. D.

    1999-01-01

    Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis. PMID:10595547

  9. Active-site cobalt(II)-substituted horse liver alcohol dehydrogenase: characterization of intermediates in the oxidation and reduction processes as a function of pH.

    PubMed

    Sartorius, C; Gerber, M; Zeppezauer, M; Dunn, M F

    1987-02-10

    Substitution of Co(II) for the catalytic site Zn(II) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, CoIIE, with characteristic Co(II) charge-transfer and d-d electronic transitions that are sensitive to the events which take place during catalysis [Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., & Dunn, M. F. (1983) Biochemistry 22, 3424-3431]. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH2OH) oxidation at pH above 7 is characterized by the formation and decay of an intermediate with lambda max = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohol, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2',2''-trifluoroethoxide ion (TFE-), CoIIE(NAD+, TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH2O-) complex, CoIIE(NAD+, RCH2O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, i.e., CoIIE(NAD+, RCH2O-) in equilibrium CoIIE(NADH, RCHO). From competition experiments, we infer that, at pH values below 5, NAD+ and alcohol form a CoIIE(NAD+, RCH2OH) ternary complex. RSSF studies carried out as a function of pH indicate that the apparent pKa values for the ionization of alcohol within the ternary complex, i.e., CoIIE(NAD+, RCH2OH) in equilibrium CoIIE(NAD+, RCH2O-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for

  10. A study of hydrogen bond effects on the oxygen, nitrogen, and hydrogen electric field gradient tensors in the active site of human dehydroepiandrosterone sulphotransferase: A density-functional theory based treatment

    NASA Astrophysics Data System (ADS)

    Astani, Elahe; Heshmati, Emran; Chen, Chun-Jung; Hadipour, Nasser L.; Shekarsaraei, Setareh

    2016-06-01

    An investigation of the density functional theory (DFT) was carried out to study the effects of the hydrogen bond (HB) interactions on 2H, 14N, and 17O electric field gradient (EFG) tensors in the active site of human dehydroepiandrosterone sulphotransferase (SULT2A1/DHEA). Natural Bonding Orbital (NBO) analysis was also performed on this active site. The results revealed that the magnitude of the quadrupole coupling constant (QCC) change at each nucleus directly depends on its contribution amount to the HB interaction.

  11. Lanthanide Metal-Organic Frameworks with Six-Coordinated Ln(III) Ions and Free Functional Organic Sites for Adsorptions and Extensive Catalytic Activities

    NASA Astrophysics Data System (ADS)

    Zhu, Yu; Zhu, Min; Xia, Li; Wu, Yunlong; Hua, Hui; Xie, Jimin

    2016-07-01

    Three chelating-amino-functionalized lanthanide metal-organic frameworks, Y-DDQ, Dy-DDQ and Eu-DDQ, were synthesized with a flexible dicarboxylate ligand based on quinoxaline (H2DDQ = N, N‧-dibenzoic acid-2,3-diaminoquinoxaline). The three-dimensional framework is constructed by the H2DDQ linkers connecting the zigzag ladders, showing a net of sra topology. In the structures, one kind of Ln(III) ions metal centers are six-coordinated and thus can potentially behave as open metal sites (OMSs), while the free chelating amino groups can act as free functional organic sites (FOSs). The N2 and Ar adsorption behaviors indicate that these Ln-DDQ exhibits stable microporous frameworks with high surface area after remove of the solvents. Owing to presence of OMSs and FOSs, these MOFs show good ability of CO2, dyes captures and Lewis acid catalyst for cyanosilylation reaction. In view of the existing FOSs in the framework, Pd NPs were immobilized onto the MOFs through graft interactions between free chelating amino groups and metal ions precursor using postsynthetic modification. The well dispersed Pd@Ln-DDQs exhibit efficient and recyclable catalytic reduction of 4-nitrophenol to 4-aminophenol, and they can also act as an excellent catalyst for Suzuki-Miyaura cross-coupling reactions with the exposed Pd NPs.

  12. Lanthanide Metal-Organic Frameworks with Six-Coordinated Ln(III) Ions and Free Functional Organic Sites for Adsorptions and Extensive Catalytic Activities

    PubMed Central

    Zhu, Yu; Zhu, Min; Xia, Li; Wu, Yunlong; Hua, Hui; Xie, Jimin

    2016-01-01

    Three chelating-amino-functionalized lanthanide metal-organic frameworks, Y-DDQ, Dy-DDQ and Eu-DDQ, were synthesized with a flexible dicarboxylate ligand based on quinoxaline (H2DDQ = N, N′-dibenzoic acid-2,3-diaminoquinoxaline). The three-dimensional framework is constructed by the H2DDQ linkers connecting the zigzag ladders, showing a net of sra topology. In the structures, one kind of Ln(III) ions metal centers are six-coordinated and thus can potentially behave as open metal sites (OMSs), while the free chelating amino groups can act as free functional organic sites (FOSs). The N2 and Ar adsorption behaviors indicate that these Ln-DDQ exhibits stable microporous frameworks with high surface area after remove of the solvents. Owing to presence of OMSs and FOSs, these MOFs show good ability of CO2, dyes captures and Lewis acid catalyst for cyanosilylation reaction. In view of the existing FOSs in the framework, Pd NPs were immobilized onto the MOFs through graft interactions between free chelating amino groups and metal ions precursor using postsynthetic modification. The well dispersed Pd@Ln-DDQs exhibit efficient and recyclable catalytic reduction of 4-nitrophenol to 4-aminophenol, and they can also act as an excellent catalyst for Suzuki-Miyaura cross-coupling reactions with the exposed Pd NPs. PMID:27431731

  13. Structure and function relationship of toxin from Chinese scorpion Buthus martensii Karsch (BmKAGAP): gaining insight into related sites of analgesic activity.

    PubMed

    Cui, Yong; Guo, Gui-Li; Ma, Lin; Hu, Nan; Song, Yong-Bo; Liu, Yan-Feng; Wu, Chun-Fu; Zhang, Jing-Hai

    2010-06-01

    In this study, an effective Escherichia coli expression system was used to study the role of residues in the antitumor-analgesic peptide from Chinese scorpion Buthus martensii Karsch (BmKAGAP). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, nine mutants of BmKAGAP were obtained by PCR. Using site-directed mutagenesis, all of these residues were individually substituted by one amino acid. These were then subjected to a circular dichroism analysis, and an analgesic activity assay in mice. This study represents a thorough mapping and elucidation of the epitopes that underlie the molecular basis of the analgesic activity. The three-dimensional structure of BmKAGAP was established by homology modeling. Our results revealed large mutant-dependent differences that indicated important roles for the studied residues. With our ongoing efforts for establishing the structure and analgesic activity relationship of BmKAGAP, we have succeeded in pinpointing which residues are important for the analgesic activity.

  14. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

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

    PubMed

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

    2012-05-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

  16. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  17. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  18. Extra-gonadal sites of estrogen biosynthesis and function

    PubMed Central

    Barakat, Radwa; Oakley, Oliver; Kim, Heehyen; Jin, Jooyoung; Ko, CheMyong Jay

    2016-01-01

    Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496] PMID:27530684

  19. HANFORD SITE WELDING PROGRAM SUCCESSFULLY PROVIDING A SINGLE SITE FUNCTION FOR USE BY MULTIPLE CONTRACTORS

    SciTech Connect

    CANNELL GR

    2009-11-19

    The Department of Energy, Richland Operations (DOE-RL) recently restructured its Hanford work scope, awarding two new contracts over the past several months for a total of three contracts to manage the sites cleanup efforts. DOE-RL met with key contractor personnel prior to and during contract transition to ensure site welding activities had appropriate oversight and maintained code compliance. The transition also provided an opportunity to establish a single site-wide function that would provide welding and materials engineering services to the Hanford site contractors: CH2M HILL Plateau Remediation Company (CHPRC); Mission Support Alliance (MSA); Washington River Protection Solutions (WRPS); and Washington Closure Hanford (WCH). Over the years, multiple and separate welding programs (amongst the several contractors) existed at the Hanford site leading to inefficiencies resulting from duplication of administrative efforts, maintenance of welding procedures, welder performance certifications, etc. The new, single program eliminates these inefficiencies. The new program, co-managed by two of the sites' new contractors, the CHPRC ('owner' of the program and responsible for construction welding services) and the MSA (provides maintenance welding services), provides more than just the traditional construction and maintenance welding services. Also provided, are welding engineering, specialty welding development/qualification for the closure of radioactive materials containers and materials evaluation/failure analysis. The following describes the new Hanford site welding program.

  20. Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure.

    PubMed

    Sakaguchi, Masayoshi; Matsushima, Yudai; Nagamine, Yusuke; Matsuhashi, Tomoki; Honda, Shotaro; Okuda, Shoi; Ohno, Misa; Sugahara, Yasusato; Shin, Yongchol; Oyama, Fumitaka; Kawakita, Masao

    2017-03-01

    Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

  1. Patterns of oligonucleotide distribution within DNA and RNA functional sites

    SciTech Connect

    Kolchanov, N.A.; Kel, A.E.; Ponomarenko, M.P.; Romachenko, A.G.; Likchachev, J.; Milanesi, L.; Lim, H.

    1993-12-31

    Patterns of short oligonucleotide distribution within DNA and RNA functional sites have been analyzed using ``Site-Video`` computer system. The group of DNA functional sites involved nucleosome binding sites, gyrase cleavage sites, promoters of E. coli and men. The group of RNA functional sites involved donor and acceptor splice sites of men, translation initiation sites of E. coli and men and translation frame shift site sites. Analysis of these samples of nucleotide sequences have been carried out by the ``Site-Video`` computer system. For each type of site specific set of patterns of oligonucleotide distribution important for the functioning and recognition have been revealed. At the same time, the number of specific patterns revealed in RNA sites was significantly higher than those in DNA sites. On the base of the results obtained, the script of functional sites for evolutionary emergency have been prompted. According to it, two types of context feature selection took place: (1) positive selection targeted to the appearance of the definite types of context features in particular regions of functional sites;and (2) negative selection targeted to the elimination of definite types of context features in particular regions of functional sites. The authors suppose that evolutionary formation of any functional site is a multistep process realized via combination of positive and negative selections. Negative selection, via fixation of a specific pattern of mutations, eliminates false signals of regulatory proteins binding with the functional site. Positive selection leads to the appearance of local context features (signals) which provide for the specificity and efficiency of the site functioning.

  2. High Precision Prediction of Functional Sites in Protein Structures

    PubMed Central

    Buturovic, Ljubomir; Wong, Mike; Tang, Grace W.; Altman, Russ B.; Petkovic, Dragutin

    2014-01-01

    We address the problem of assigning biological function to solved protein structures. Computational tools play a critical role in identifying potential active sites and informing screening decisions for further lab analysis. A critical parameter in the practical application of computational methods is the precision, or positive predictive value. Precision measures the level of confidence the user should have in a particular computed functional assignment. Low precision annotations lead to futile laboratory investigations and waste scarce research resources. In this paper we describe an advanced version of the protein function annotation system FEATURE, which achieved 99% precision and average recall of 95% across 20 representative functional sites. The system uses a Support Vector Machine classifier operating on the microenvironment of physicochemical features around an amino acid. We also compared performance of our method with state-of-the-art sequence-level annotator Pfam in terms of precision, recall and localization. To our knowledge, no other functional site annotator has been rigorously evaluated against these key criteria. The software and predictive models are incorporated into the WebFEATURE service at http://feature.stanford.edu/wf4.0-beta. PMID:24632601

  3. [Natural organic compounds that affect to microtubule functions: syntheses and structure-activity relationships of combretastatins, curacin A and their analogs as the colchicine-site ligands on tubulin].

    PubMed

    Iwasaki, S; Shirai, R

    2000-10-01

    Microtubules (MT) are cylindrical polymers of the protein tubulin (TN) alpha, beta-heterodimer, and are known to be the main component of spindles in mitotic apparatus of eucaryotic cells. MT are also involved in many other basic and essential cell functions. There are a number of natural and synthetic compounds that interfere with MT function to cause the mitotic arrest of eucaryotic cells. Such antimitotic agents show a broad biological activity, and can be used for medicinal and agrochemical purposes. On the other hand, they are important also as the biochemical tools for understanding the dynamics of MT network. Most of such antimitotic agents, with a few exceptions, bind to beta-TN. Among them, colchicine (CLC), vinblastine and taxol have played major roles in practical uses as well as in biochemical studies of MT functions. They all bind to beta-TN but their binding sites are different. We have worked on a variety of antimitotic agents that bind to either of colchicine-site, vinblastine-site and taxol-site, in discovery, structures, biological actions and/or interactions with TN. In this paper, the results of our studies on CLC-site ligands were summarized; (1) synthetic analogs of combretastatin A-4 (CBS A-4), isolated as a cytotoxic compound produced by a species of South African tree Combretum caffrum, (2) curacin A (CU-A), a cytotoxic metabolite of a marine cyanobacteria Lyngbya majuscula, and its related compounds. Interactions of these compounds with TN were studied and structure-activity relationships of these two classes of compounds were discussed.

  4. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  5. Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes

    PubMed Central

    Jack, Benjamin R.; Meyer, Austin G.; Echave, Julian; Wilke, Claus O.

    2016-01-01

    Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein–protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes. PMID:27138088

  6. Text Mining Improves Prediction of Protein Functional Sites

    PubMed Central

    Cohn, Judith D.; Ravikumar, Komandur E.

    2012-01-01

    We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions. PMID:22393388

  7. Plasmodium subtilisin-like protease 1 (SUB1): Insights into the active-site structure, specificity and function of a pan-malaria drug target

    PubMed Central

    Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J.

    2012-01-01

    Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop ‘pan-reactive’ drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. PMID:22543039

  8. Quantum cluster size and solvent polarity effects on the geometries and Mössbauer properties of the active site model for ribonucleotide reductase intermediate X: a density functional theory study.

    PubMed

    Han, Wen-Ge; Noodleman, Louis

    2010-03-01

    In studying the properties of metalloproteins using ab initio quantum mechanical methods, one has to focus on the calculations on the active site. The bulk protein and solvent environment is often neglected, or is treated as a continuum dielectric medium with a certain dielectric constant. The size of the quantum cluster of the active site chosen for calculations can vary by including only the first-shell ligands which are directly bound to the metal centers, or including also the second-shell residues which are adjacent to and normally have H-bonding interactions with the first-shell ligands, or by including also further hydrogen bonding residues. It is not well understood how the size of the quantum cluster and the value of the dielectric constant chosen for the calculations will influence the calculated properties. In this paper, we have studied three models (A, B, and C) of different sizes for the active site of the ribonucleotide reductase intermediate X, using density functional theory (DFT) OPBE functional with broken-symmetry methodology. Each model is studied in gas-phase and in the conductor-like screening (COSMO) solvation model with different dielectric constants ε = 4, 10, 20, and 80, respectively. All the calculated Fe-ligand geometries, Heisenberg J coupling constants, and the Mössbauer isomer shifts, quadrupole splittings, and the (57)Fe, (1)H, and (17)O hyperfine tensors are compared. We find that the calculated isomer shifts are very stable. They are virtually unchanged with respect to the size of the cluster and the dielectric constant of the environment. On the other hand, certain Fe-ligand distances are sensitive to both the size of the cluster and the value of ε. ε = 4, which is normally used for the protein environment, appears too small when studying the diiron active site geometry with only the first-shell ligands as seen by comparisons with larger models.

  9. Biomimetic oxidation studies. 9. Mechanistic aspects of the oxidation of alcohols with functional,active site methane monooxygenase enzyme models in aqueous solution

    SciTech Connect

    Rabion, A. ||; Chen, S.; Wang, J.; Buchanan, R.M.; Seris, J.L.; Fish, R.H. |

    1995-12-13

    The syntheses of biomimetic complexes that mimic the major structural features of the hydroxylase component of methane monooxygenase enzyme (MMO) and, more importantly, that provide similar alkane functionalization activity, in the presence of an oxidant, have been of great interest to the discipline of bioinorganic chemistry. In this communication, we will demonstrate the feasibility of conducting biomimetic oxidation studies in H{sub 2}O with soluble substrates, i.e., alcohols (cyclohexanol, benzyl alcohol), using H{sub 2}O-stable MMO mimics at pH 4.2, and the oxidant, tert-butyl hydroperoxide (TBHP). Both the Mitusunobu procedure and the mesylate displacement reaction proceeded with complete inversion of the stereo-center and provided optically pure penultimate intermediate (>99.9% ee). The synthesis was completed by reduction of the nitro group under standard conditions to deliver LY300164 in 87%. In summary, we have developed an efficient and environmentally benign synthesis of the 5H-2,3-benzodiazepine LY300164 that provides the optically pure compound in 51% overall yield. Intramolecular hydrazone alkylation led to a remarkably facile and selective formation of the benzodiazepine. Furthermore, the application of resins to whole-cell-based biotransformations should find general utility for similar reactions that are complicated by component inhibition and product isolation. 11 refs., 1 fig.

  10. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

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

  11. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

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

  12. Density functional theory calculations on the active site of biotin synthase: mechanism of S transfer from the Fe(2)S(2) cluster and the role of 1st and 2nd sphere residues.

    PubMed

    Rana, Atanu; Dey, Subal; Agrawal, Amita; Dey, Abhishek

    2015-10-01

    Density functional theory (DFT) calculations are performed on the active site of biotin synthase (BS) to investigate the sulfur transfer from the Fe(2)S(2) cluster to dethiobiotin (DTB). The active site is modeled to include both the 1st and 2nd sphere residues. Molecular orbital theory considerations and calculation on smaller models indicate that only an S atom (not S²⁻) transfer from an oxidized Fe(2)S(2) cluster leads to the formation of biotin from the DTB using two adenosyl radicals generated from S-adenosyl-L-methionine. The calculations on larger protein active site model indicate that a 9-monothiobiotin bound reduced cluster should be an intermediate during the S atom insertion from the Fe(2)S(2) cluster consistent with experimental data. The Arg260 bound to Fe1, being a weaker donor than cysteine bound to Fe(2), determines the geometry and the electronic structure of this intermediate. The formation of this intermediate containing the C9-S bond is estimated to have a ΔG(≠) of 17.1 kcal/mol while its decay by the formation of the 2nd C6-S bond is calculated to have a ΔG(≠) of 29.8 kcal/mol, i.e. the 2nd C-S bond formation is calculated to be the rate determining step in the cycle and it leads to the decay of the Fe(2)S(2) cluster. Significant configuration interaction (CI), present in these transition states, helps lower the barrier of these reactions by ~30-25 kcal/mol relative to a hypothetical outer-sphere reaction. The conserved Phe285 residue near the Fe(2)S(2) active site determines the stereo selectivity at the C6 center of this radical coupling reaction. Reaction mechanism of BS investigated using DFT calculations. Strong CI and the Phe285 residue control the kinetic rate and stereochemistry of the product.

  13. De novo active sites for resurrected Precambrian enzymes

    PubMed Central

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

    2017-01-01

    Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering. PMID:28719578

  14. De novo active sites for resurrected Precambrian enzymes.

    PubMed

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

    2017-07-18

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

  15. De novo active sites for resurrected Precambrian enzymes

    NASA Astrophysics Data System (ADS)

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

    2017-07-01

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

  16. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  17. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  18. Functional constraints on adaptive evolution of protein ubiquitination sites

    PubMed Central

    Lu, Liang; Li, Yang; Liu, Zhongyang; Liang, Fengji; Guo, Feifei; Yang, Shuai; Wang, Dan; He, Yangzhige; Xiong, Jianghui; Li, Dong; He, Fuchu

    2017-01-01

    It is still unclear whether there exist functional constraints on the evolution of protein ubiquitination sites, because most previous studies regarded all protein ubiquitination sites as a whole or only focused on limited structural properties. We tried to clarify the relation between functional constraints and ubiquitination sites evolution. We investigated the evolutionary conservation of human ubiquitination sites in a broad evolutionary scale from G. gorilla to S. pombe, and we found that in organisms originated after the divergence of vertebrate, ubiquitination sites are more conserved than their flanking regions, while the opposite tendency is observed before this divergence time. By grouping the ubiquitination proteins into different functional categories, we confirm that many functional constraints like certain molecular functions, protein tissue expression specificity and protein connectivity in protein-protein interaction network enhance the evolutionary conservation of ubiquitination sites. Furthermore, by analyzing the gains of ubiquitination sites at different divergence time and their functional characters, we validate that the emergences of ubiquitination sites at different evolutionary time were also affected by the uncovered functional constraints. The above results suggest that functional constraints on the adaptive evolution of ubiquitination sites increase the opportunity for ubiquitination to synthetically regulate various cellular and developmental processes during evolution. PMID:28054638

  19. Localizing the site of magnetic brain stimulation by functional MRI.

    PubMed

    Terao, Y; Ugawa, Y; Sakai, K; Miyauchi, S; Fukuda, H; Sasaki, Y; Takino, R; Hanajima, R; Furubayashi, T; Pütz, B; Kanazawa, I

    1998-07-01

    In order to locate the site of action of transcranial magnetic stimulation (TMS) within the human motor cortices, we investigated how the optimal positions for evoking motor responses over the scalp corresponded to the hand and leg primary-motor areas. TMS was delivered with a figure-8 shaped coil over each point of a grid system constructed on the skull surface, each separated by 1 cm, to find the optimal site for obtaining motor-evoked potentials (MEPs) in the contralateral first dorsal interosseous (FDI) and tibialis anterior (TA) muscles. Magnetic resonance imaging scans of the brain were taken for each subject with markers placed over these sites, the positions of which were projected onto the cortical region just beneath. On the other hand, cortical areas where blood flow increased during finger tapping or leg movements were identified on functional magnetic resonance images (fMRI), which should include the hand and leg primary-motor areas. The optimal location for eliciting MEPs in FDI, regardless of their latency, lay just above the bank of the precentral gyrus, which coincided with the activated region during finger tapping in fMRI studies. The direction of induced current preferentially eliciting MEPs with the shortest latency in each subject was nearly perpendicular to the course of the precentral gyrus at this position. The optimal site for evoking motor responses in TA was also located just above the activated area during leg movements identified within the anterior portion of the paracentral lobule. The results suggest that, for magnetic stimulation, activation occurs in the primary hand and leg motor area (Brodmann area 4), which is closest in distance to the optimal scalp position for evoking motor responses.

  20. Functional insight into Maelstrom in the germline piRNA pathway: a unique domain homologous to the DnaQ-H 3'–5' exonuclease, its lineage-specific expansion/loss and evolutionarily active site switch

    PubMed Central

    Zhang, Dapeng; Xiong, Huiling; Shan, Jufang; Xia, Xuhua; Trudeau, Vance L

    2008-01-01

    Abstract Maelstrom (MAEL) plays a crucial role in a recently-discovered piRNA pathway; however its specific function remains unknown. Here a novel MAEL-specific domain characterized by a set of conserved residues (Glu-His-His-Cys-His-Cys, EHHCHC) was identified in a broad range of species including vertebrates, sea squirts, insects, nematodes, and protists. It exhibits ancient lineage-specific expansions in several species, however, appears to be lost in all examined teleost fish species. Functional involvement of MAEL domains in DNA- and RNA-related processes was further revealed by its association with HMG, SR-25-like and HDAC_interact domains. A distant similarity to the DnaQ-H 3'–5' exonuclease family with the RNase H fold was discovered based on the evidence that all MAEL domains adopt the canonical RNase H fold; and several protist MAEL domains contain the conserved 3'–5' exonuclease active site residues (Asp-Glu-Asp-His-Asp, DEDHD). This evolutionary link together with structural examinations leads to a hypothesis that MAEL domains may have a potential nuclease activity or RNA-binding ability that may be implicated in piRNA biogenesis. The observed transition of two sets of characteristic residues between the ancestral DnaQ-H and the descendent MAEL domains may suggest a new mode for protein function evolution called "active site switch", in which the protist MAEL homologues are the likely evolutionary intermediates due to harboring the specific characteristics of both 3'–5' exonuclease and MAEL domains. Reviewers This article was reviewed by L Aravind, Wing-Cheong Wong and Frank Eisenhaber. For the full reviews, please go to the Reviewers' Comments section. PMID:19032786

  1. Functional insight into Maelstrom in the germline piRNA pathway: a unique domain homologous to the DnaQ-H 3'-5' exonuclease, its lineage-specific expansion/loss and evolutionarily active site switch.

    PubMed

    Zhang, Dapeng; Xiong, Huiling; Shan, Jufang; Xia, Xuhua; Trudeau, Vance L

    2008-11-25

    Maelstrom (MAEL) plays a crucial role in a recently-discovered piRNA pathway; however its specific function remains unknown. Here a novel MAEL-specific domain characterized by a set of conserved residues (Glu-His-His-Cys-His-Cys, EHHCHC) was identified in a broad range of species including vertebrates, sea squirts, insects, nematodes, and protists. It exhibits ancient lineage-specific expansions in several species, however, appears to be lost in all examined teleost fish species. Functional involvement of MAEL domains in DNA- and RNA-related processes was further revealed by its association with HMG, SR-25-like and HDAC_interact domains. A distant similarity to the DnaQ-H 3'-5' exonuclease family with the RNase H fold was discovered based on the evidence that all MAEL domains adopt the canonical RNase H fold; and several protist MAEL domains contain the conserved 3'-5' exonuclease active site residues (Asp-Glu-Asp-His-Asp, DEDHD). This evolutionary link together with structural examinations leads to a hypothesis that MAEL domains may have a potential nuclease activity or RNA-binding ability that may be implicated in piRNA biogenesis. The observed transition of two sets of characteristic residues between the ancestral DnaQ-H and the descendent MAEL domains may suggest a new mode for protein function evolution called "active site switch", in which the protist MAEL homologues are the likely evolutionary intermediates due to harboring the specific characteristics of both 3'-5' exonuclease and MAEL domains.

  2. Functional mapping of the lectin activity site on the β-prism domain of vibrio cholerae cytolysin: implications for the membrane pore-formation mechanism of the toxin.

    PubMed

    Rai, Anand Kumar; Paul, Karan; Chattopadhyay, Kausik

    2013-01-18

    Vibrio cholerae cytolysin (VCC) is a prominent member in the family of β-barrel pore-forming toxins. It induces lysis of target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the β-Prism domain, we demonstrate that the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the β-Prism domain's lectin activity, implicated in the process of membrane pore formation by VCC.

  3. The single site Green s function and Krein s theorem

    SciTech Connect

    Wang, Yang; Stocks, George Malcolm; Faulkner, John Sam

    2014-01-01

    An important step in electronic structure calculations using multiple-scattering theory is obtaining the density of states for the central site from the Green s function for that site. We have found that the Krein s spectral displacement function for the central site contributes significantly to the understanding of these calculations. We argue that these insights can lead to improvements in the robustness of MST electronic structure codes without negatively impacting their performance.

  4. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  5. Prediction of functional sites in proteins using conserved functional group analysis.

    PubMed

    Innis, C Axel; Anand, A Prem; Sowdhamini, R

    2004-04-02

    A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects.

  6. 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.

  7. Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues.

    PubMed

    Saadat, D; Harrison, D H

    1998-07-14

    Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.

  8. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  9. 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.

  10. Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation

    PubMed Central

    Peng, R; Tong, J-S; Li, H; Yue, B; Zou, F; Yu, J; Zhang, L

    2013-01-01

    Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis. PMID:23392123

  11. Metric learning for enzyme active-site search

    PubMed Central

    Kato, Tsuyoshi; Nagano, Nozomi

    2010-01-01

    Motivation: Finding functionally analogous enzymes based on the local structures of active sites is an important problem. Conventional methods use templates of local structures to search for analogous sites, but their performance depends on the selection of atoms for inclusion in the templates. Results: The automatic selection of atoms so that site matches can be discriminated from mismatches. The algorithm provides not only good predictions, but also some insights into which atoms are important for the prediction. Our experimental results suggest that the metric learning automatically provides more effective templates than those whose atoms are selected manually. Availability: Online software is available at http://www.net-machine.net/∼kato/lpmetric1/ Contact: kato-tsuyoshi@k.u-tokyo.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20870642

  12. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

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

    2016-01-01

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

  13. Examining Search Functions of EAD Finding Aids Web Sites

    ERIC Educational Resources Information Center

    Zhou, Xiaomu

    2006-01-01

    This article examined the search functions for all individual EAD Web sites listed on the Library of Congress Web site in 2003. In particular, the type of search engine, search modes, options for searching, search results display, search feedback, and other features of the search systems were studied. The data analysis suggests that there have…

  14. Examining Search Functions of EAD Finding Aids Web Sites

    ERIC Educational Resources Information Center

    Zhou, Xiaomu

    2006-01-01

    This article examined the search functions for all individual EAD Web sites listed on the Library of Congress Web site in 2003. In particular, the type of search engine, search modes, options for searching, search results display, search feedback, and other features of the search systems were studied. The data analysis suggests that there have…

  15. Conserved Functional Motifs and Homology Modeling to Predict Hidden Moonlighting Functional Sites

    PubMed Central

    Wong, Aloysius; Gehring, Chris; Irving, Helen R.

    2015-01-01

    Moonlighting functional centers within proteins can provide them with hitherto unrecognized functions. Here, we review how hidden moonlighting functional centers, which we define as binding sites that have catalytic activity or regulate protein function in a novel manner, can be identified using targeted bioinformatic searches. Functional motifs used in such searches include amino acid residues that are conserved across species and many of which have been assigned functional roles based on experimental evidence. Molecules that were identified in this manner seeking cyclic mononucleotide cyclases in plants are used as examples. The strength of this computational approach is enhanced when good homology models can be developed to test the functionality of the predicted centers in silico, which, in turn, increases confidence in the ability of the identified candidates to perform the predicted functions. Computational characterization of moonlighting functional centers is not diagnostic for catalysis but serves as a rapid screening method, and highlights testable targets from a potentially large pool of candidates for subsequent in vitro and in vivo experiments required to confirm the functionality of the predicted moonlighting centers. PMID:26106597

  16. Site-site direct correlation functions for three popular molecular models of liquid water.

    PubMed

    Zhao, Shuangliang; Liu, Yu; Liu, Honglai; Wu, Jianzhong

    2013-08-14

    Direct correlation functions (DCFs) play a pivotal role in the applications of classical density functional theory (DFT) to addressing the thermodynamic properties of inhomogeneous systems beyond the local-density or mean-field approximations. Whereas numerous studies have been dedicated to the radial distribution functions of liquid water--the most important solvent on earth, relatively little attention has been given to the site-site DCFs. The water DCFs are long-ranged and difficult to calculate directly by simulation, and the predictions from conventional liquid-state theories have been rarely calibrated. Here we report a computational procedure for accurate evaluation of the site-site DCFs of liquid water based on three popular molecular models (viz., SPC, SPC∕E, and TIP3P). The numerical results provide a benchmark for calibration of conventional liquid-state theories and fresh insights into development of new DFT methods. We show that: (1) the long-range behavior of the site-site DCFs depends on both the molecular model and the thermodynamic condition; (2) the asymptotic limit of DCFs at large distance does not follow the mean-spherical approximation (MSA); (3) individual site-site DCFs are long ranged (~40 nm) but a summation of all DCF pairs exhibits only short-range behavior (~1 nm or a few water diameters); (4) the site-site bridge correlation functions behave as the DCFs, i.e., they are also long-ranged while the summation of all bridge correlation functions is short ranged. Our analytical and numerical analyses of the DCFs provide some simple strategies for possible improvement of the numerical performance of conventional liquid-state theories.

  17. Context-dependent function of "GATA switch" sites in vivo.

    PubMed

    Snow, Jonathan W; Trowbridge, Jennifer J; Johnson, Kirby D; Fujiwara, Tohru; Emambokus, Nikla E; Grass, Jeffrey A; Orkin, Stuart H; Bresnick, Emery H

    2011-05-05

    Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the -2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring -1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the -2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

  18. A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

    PubMed

    Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena; Davies, Christopher; Nicholas, Robert A

    2012-04-03

    Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

  19. T-Loop Phosphorylated Cdk9 Localizes to Nuclear Speckle Domains Which May Serve as Sites of Active P-TEFb Function and Exchange Between the Brd4 and 7SK/HEXIM1 Regulatory Complexes

    PubMed Central

    Dow, Eugene C.; Liu, Hongbing; Rice, Andrew P.

    2010-01-01

    P-TEFb functions to induce the elongation step of RNA polymerase II transcription by phosphorylating the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Core P-TEFb is comprised of Cdk9 and a cyclin regulatory subunit, with Cyclin T1 being the predominant Cdk9-associated cyclin. The kinase activity of P-TEFb is dependent on phosphorylation of the Thr186 residue located within the T-loop domain of the Cdk9 subunit. Here, we used immunofluorescence deconvolution microscopy to examine the subcellular distribution of phospho-Thr186 Cdk9/Cyclin T1 P-TEFb heterodimers. We found that phospho-Thr186 Cdk9 displays a punctate distribution throughout the non-nucleolar nucleoplasm and it co-localizes with Cyclin T1 almost exclusively within nuclear speckle domains. Phospho-Thr186 Cdk9 predominantly co-localized with the hyperphosphorylated forms of RNA polymerase II. Transient expression of kinase-defective Cdk9 mutants revealed that neither is Thr186 phosphorylation or kinase activity required for Cdk9 speckle localization. Lastly, both the Brd4 and HEXIM1 proteins interact with P-TEFb at or very near speckle domains and treatment of cells with the Cdk9 inhibitor flavopiridol alters this distribution. These results indicate that the active form of P-TEFb resides in nuclear speckles and raises the possibility that speckles are sites of P-TEFb function and exchange between negative and positive P-TEFb regulatory complexes. PMID:20201073

  20. Exon grafting yields a "two active-site" lysozyme.

    PubMed

    Li, Shuang; Li, Bin; Fei, Yanqun; Jiang, Dazhi; Sheng, Yongjie; Sun, Yanhong; Zhang, Jin

    2007-07-13

    The design of enzymes with enhanced stability and activity has long been a goal in protein engineering. We report a strategy to engineer an additional active site for human lysozyme, grafted the entire human lysozyme exon 2, which encodes the catalytically competent domain, into the gene at a position corresponding to an exposed loop region in the translated protein. Exon 2 grafting created a novel lysozyme with twice the activity of the wild type enzyme, equal activity came from each of the two active sites. We dissected the contributions of each active site using site-directed mutagenesis of the catalytic doublets of (E35A/D53A), circular dichroism, fluorescence spectra, and molecular modeling. Temperature and pH stability of the "two active-site" enzyme were similar to those of wild-type lysozyme. Thus, we provide a novel strategy for engineering the active site of enzymes.

  1. Density functional theory calculations on the mononuclear non-heme iron active site of Hmd hydrogenase: role of the internal ligands in tuning external ligand binding and driving H2 heterolysis.

    PubMed

    Dey, Abhishek

    2010-10-06

    DFT calculations on active-site models of the non-heme Fe site of Hmd hydrogenase are reported. Binding of several biologically relevant ligands (e.g., CN(-), CO, H(-), H(2), and O(2)) to the active site of Hmd was investigated using a method that reproduced the geometric and vibrational properties of the resting site. The results indicate that this neutral ferrous active site has higher affinity toward anionic ligands (e.g., H(-) and CN(-)) than π-acidic ligands (e.g., CO and O(2)). Natural population analysis and molecular orbital analysis revealed that this is due to extensive delocalization of electron density into the low-lying unoccupied orbitals of the CO, acyl, and pyridinol ligands present in the active site. In addition to normal d-π back-bonding, metal 3d orbital-mediated charge transfer from occupied ligand orbitals to the unoccupied orbitals of the internal ligands was observed. This charge transfer leads to systematic variations in the experimentally observed C-O stretching frequencies. Protonation of the thiolate ligand present in the active site significantly enhances these anion ligand binding affinities. In fact, the calculated vibrational frequencies indicate that CN(-) binding is possibly associated with protonation of the thiolate ligand. The high affinity for binding of the anionic H(-) ligand (where 81% of the electron density of H(-) is delocalized into the active site) is calculated to play a dominating role in the H-H bond heterolysis step during catalysis. The binding energies of these ligands relative to the substrate, H(2), highlight the importance of a proposed structural reorganization during catalysis.

  2. The Dynamic Dielectric at a Brain Functional Site and an EM Wave Approach to Functional Brain Imaging

    PubMed Central

    Li, X. P.; Xia, Q.; Qu, D.; Wu, T. C.; Yang, D. G.; Hao, W. D.; Jiang, X.; Li, X. M.

    2014-01-01

    Functional brain imaging has tremendous applications. The existing methods for functional brain imaging include functional Magnetic Resonant Imaging (fMRI), scalp electroencephalography (EEG), implanted EEG, magnetoencephalography (MEG) and Positron Emission Tomography (PET), which have been widely and successfully applied to various brain imaging studies. To develop a new method for functional brain imaging, here we show that the dielectric at a brain functional site has a dynamic nature, varying with local neuronal activation as the permittivity of the dielectric varies with the ion concentration of the extracellular fluid surrounding neurons in activation. Therefore, the neuronal activation can be sensed by a radiofrequency (RF) electromagnetic (EM) wave propagating through the site as the phase change of the EM wave varies with the permittivity. Such a dynamic nature of the dielectric at a brain functional site provides the basis for an RF EM wave approach to detecting and imaging neuronal activation at brain functional sites, leading to an RF EM wave approach to functional brain imaging. PMID:25367217

  3. The dynamic dielectric at a brain functional site and an EM wave approach to functional brain imaging.

    PubMed

    Li, X P; Xia, Q; Qu, D; Wu, T C; Yang, D G; Hao, W D; Jiang, X; Li, X M

    2014-11-04

    Functional brain imaging has tremendous applications. The existing methods for functional brain imaging include functional Magnetic Resonant Imaging (fMRI), scalp electroencephalography (EEG), implanted EEG, magnetoencephalography (MEG) and Positron Emission Tomography (PET), which have been widely and successfully applied to various brain imaging studies. To develop a new method for functional brain imaging, here we show that the dielectric at a brain functional site has a dynamic nature, varying with local neuronal activation as the permittivity of the dielectric varies with the ion concentration of the extracellular fluid surrounding neurons in activation. Therefore, the neuronal activation can be sensed by a radiofrequency (RF) electromagnetic (EM) wave propagating through the site as the phase change of the EM wave varies with the permittivity. Such a dynamic nature of the dielectric at a brain functional site provides the basis for an RF EM wave approach to detecting and imaging neuronal activation at brain functional sites, leading to an RF EM wave approach to functional brain imaging.

  4. Prediction of Functional Sites Based on the Fuzzy Oil Drop Model

    PubMed Central

    Bryliński, Michał; Prymula, Katarzyna; Jurkowski, Wiktor; Kochańczyk, Marek; Stawowczyk, Ewa; Konieczny, Leszek; Roterman, Irena

    2007-01-01

    A description of many biological processes requires knowledge of the 3-D structure of proteins and, in particular, the defined active site responsible for biological function. Many proteins, the genes of which have been identified as the result of human genome sequencing, and which were synthesized experimentally, await identification of their biological activity. Currently used methods do not always yield satisfactory results, and new algorithms need to be developed to recognize the localization of active sites in proteins. This paper describes a computational model that can be used to identify potential areas that are able to interact with other molecules (ligands, substrates, inhibitors, etc.). The model for active site recognition is based on the analysis of hydrophobicity distribution in protein molecules. It is shown, based on the analyses of proteins with known biological activity and of proteins of unknown function, that the region of significantly irregular hydrophobicity distribution in proteins appears to be function related. PMID:17530916

  5. 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.

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

    SciTech Connect

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

    2015-03-09

    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. 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. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.

  7. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.

    PubMed Central

    Bode, W.; Turk, D.; Karshikov, A.

    1992-01-01

    Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70

  8. [Physical activity and brain function].

    PubMed

    Kempermann, G

    2012-06-01

    Physical activity has direct and indirect effects on brain function in health and disease. Findings demonstrating that physical activity improves cognitive and non-cognitive functions and is preventive for several neuropsychiatric disorders have attracted particular interest. This short review focuses on sports and physical exercise in normal brain function and summarizes which mechanisms might underlie the observed effects, which methodological problems exist, which relationships exist to concepts of plasticity and neural reserves and what evolutionary relevance the initially surprising finding that physical exercise is good for the brain has.

  9. Structure-based Methods for Computational Protein Functional Site Prediction

    PubMed Central

    Dukka, B KC

    2013-01-01

    Due to the advent of high throughput sequencing techniques and structural genomic projects, the number of gene and protein sequences has been ever increasing. Computational methods to annotate these genes and proteins are even more indispensable. Proteins are important macromolecules and study of the function of proteins is an important problem in structural bioinformatics. This paper discusses a number of methods to predict protein functional site especially focusing on protein ligand binding site prediction. Initially, a short overview is presented on recent advances in methods for selection of homologous sequences. Furthermore, a few recent structural based approaches and sequence-and-structure based approaches for protein functional sites are discussed in details. PMID:24688745

  10. Characterization of the active sites in decay-accelerating factor.

    PubMed

    Kuttner-Kondo, L A; Mitchell, L; Hourcade, D E; Medof, M E

    2001-08-15

    Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.

  11. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  12. 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.

  13. Structural characterization of single nucleotide variants at ligand binding sites and enzyme active sites of human proteins

    PubMed Central

    Yamada, Kazunori D.; Nishi, Hafumi; Nakata, Junichi; Kinoshita, Kengo

    2016-01-01

    Functional sites on proteins play an important role in various molecular interactions and reactions between proteins and other molecules. Thus, mutations in functional sites can severely affect the overall phenotype. Progress of genome sequencing projects has yielded a wealth of information on single nucleotide variants (SNVs), especially those with less than 1% minor allele frequency (rare variants). To understand the functional influence of genetic variants at a protein level, we investigated the relationship between SNVs and protein functional sites in terms of minor allele frequency and the structural position of variants. As a result, we observed that SNVs were less abundant at ligand binding sites, which is consistent with a previous study on SNVs and protein interaction sites. Additionally, we found that non-rare variants tended to be located slightly apart from enzyme active sites. Examination of non-rare variants revealed that most of the mutations resulted in moderate changes of the physico-chemical properties of amino acids, suggesting the existence of functional constraints. In conclusion, this study shows that the mapping of genetic variants on protein structures could be a powerful approach to evaluate the functional impact of rare genetic variations. PMID:27924270

  14. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    PubMed

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy.

  15. First Principles Computational Study of the Active Site of Arginase

    SciTech Connect

    Ivanov, Ivaylo; Klien, Micheal

    2004-01-14

    Ab initio density functional theory (DFT) methods were used to investigate the structural features of the active site of the binuclear enzyme rat liver arginase. Special emphasis was placed on the crucial role of the second shell ligand interactions. These interactions were systematically studied by performing calculations on models of varying size. It was determined that a water molecule, and not hydroxide, is the bridging exogenous ligand. The carboxylate ligands facilitate the close approach of the Mn (II) ions by attenuating the metal-metal electrostatic repulsion. Of the two metals, MnA was shown to carry a larger positive charge. Analysis of the electronic properties of the active site revealed that orbitals involving the terminal Asp234 residue, as well as the flexible -1,1 bridging Asp232, lie at high energies, suggesting weaker coordination. This is reflected in certain structural variability present in our models and is also consistent with recent experimental findings. Finally, implications of our findings for the biological function of the enzyme are delineated.

  16. Melanocortin MC₄ receptor expression sites and local function.

    PubMed

    Siljee-Wong, Jacqueline E

    2011-06-11

    The melanocortin MC(4) receptor plays an important role in energy metabolism, but also affects blood pressure, heart rate and erectile function. Localization of the receptors that fulfill these distinct roles is only partially known. Mapping of the melanocortin MC(4) receptor has been stymied by the absence of a functional antibody. Several groups have examined mRNA expression of the melanocortin MC(4) receptor in the rodent brain and transgenic approaches have also been utilized to visualize melanocortin MC(4) receptor expression sites within the brain. Ligand expression and binding studies have provided additional information on the areas of the brain where this elusive receptor is functionally expressed. Finally, microinjection of melanocortin MC(4) receptor ligands in specific nuclei has further served to elucidate the function of melanocortin MC(4) receptors in these nuclei. These combined approaches have helped link the anatomy and function of this receptor, such as the role of paraventricular hypothalamic nucleus melanocortin MC(4) receptor in the regulation of food intake. Intriguingly, however, numerous expression-sites have been identified that have not been linked to a specific receptor function such as those along the optic tract and olfactory tubercle. Further research is needed to clarify the function of the melanocortin MC(4) receptor at these sites.

  17. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2013-10-01

    mice and mice transfused with Syk inhibitor-treated platelets . Platelet lodging was remarkably decreased in lungs of mice transfused with Syk...AD_________________ Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet ...30September2012–29September2013 4. TITLE AND SUBTITLE Complement Activation Alters Platelet Function 5a. CONTRACT NUMBER W81XWH-12-1-0523 5b. GRANT NUMBER

  18. Activation barriers of oxygen transformation at the active site of [FeFe] hydrogenases.

    PubMed

    Finkelmann, Arndt R; Stiebritz, Martin T; Reiher, Markus

    2014-11-17

    Oxygen activation at the active sites of [FeFe] hydrogenases has been proposed to be the initial step of irreversible oxygen-induced inhibition of these enzymes. On the basis of a first theoretical study into the thermodynamics of O2 activation [Inorg. Chem. 2009, 48, 7127] we here investigate the kinetics of possible reaction paths at the distal iron atom of the active site by means of density functional theory. A sequence of steps is proposed to either form a reactive oxygen species (ROS) or fully reduce O2 to water. In this reaction cascade, two branching points are identified where water formation directly competes with harmful oxygen activation reactions. The latter are water formation by O-O bond cleavage of a hydrogen peroxide-bound intermediate competing with H2O2 dissociation and CO2 formation by a putative iron-oxo species competing with protonation of the iron-oxo species to form a hydroxyo ligand. Furthermore, we show that proton transfer to activated oxygen is fast and that proton supply to the active site is vital to prevent ROS dissociation. If sufficiently many reduction equivalents are available, oxygen activation reactions are accelerated, and oxygen reduction to water becomes possible.

  19. 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.

  20. 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.

  1. 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.

  2. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

    PubMed Central

    Lim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, Inchan

    2016-01-01

    Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other. PMID:28004799

  3. 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.

  4. 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

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

    DOE PAGES

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; ...

    2015-03-09

    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. 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 themore » 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. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.« less

  6. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    PubMed

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

  7. Predicting active site residue annotations in the Pfam database.

    PubMed

    Mistry, Jaina; Bateman, Alex; Finn, Robert D

    2007-08-09

    Approximately 5% of Pfam families are enzymatic, but only a small fraction of the sequences within these families (<0.5%) have had the residues responsible for catalysis determined. To increase the active site annotations in the Pfam database, we have developed a strict set of rules, chosen to reduce the rate of false positives, which enable the transfer of experimentally determined active site residue data to other sequences within the same Pfam family. We have created a large database of predicted active site residues. On comparing our active site predictions to those found in UniProtKB, Catalytic Site Atlas, PROSITE and MEROPS we find that we make many novel predictions. On investigating the small subset of predictions made by these databases that are not predicted by us, we found these sequences did not meet our strict criteria for prediction. We assessed the sensitivity and specificity of our methodology and estimate that only 3% of our predicted sequences are false positives. We have predicted 606110 active site residues, of which 94% are not found in UniProtKB, and have increased the active site annotations in Pfam by more than 200 fold. Although implemented for Pfam, the tool we have developed for transferring the data can be applied to any alignment with associated experimental active site data and is available for download. Our active site predictions are re-calculated at each Pfam release to ensure they are comprehensive and up to date. They provide one of the largest available databases of active site annotation.

  8. Functional interaction of nitrogenous organic bases with cytochrome P450: a critical assessment and update of substrate features and predicted key active-site elements steering the access, binding, and orientation of amines.

    PubMed

    Hlavica, Peter

    2006-04-01

    The widespread use of nitrogenous organic bases as environmental chemicals, food additives, and clinically important drugs necessitates precise knowledge about the molecular principles governing biotransformation of this category of substrates. In this regard, analysis of the topological background of complex formation between amines and P450s, acting as major catalysts in C- and N-oxidative attack, is of paramount importance. Thus, progress in collaborative investigations, combining physico-chemical techniques with chemical-modification as well as genetic engineering experiments, enables substantiation of hypothetical work resulting from the design of pharmacophores or homology modelling of P450s. Based on a general, CYP2D6-related construct, the majority of prospective amine-docking residues was found to cluster near the distal heme face in the six known SRSs, made up by the highly variant helices B', F and G as well as the N-terminal portion of helix C and certain beta-structures. Most of the contact sites examined show a frequency of conservation < 20%, hinting at the requirement of some degree of conformational versatility, while a limited number of amino acids exhibiting a higher level of conservation reside close to the heme core. Some key determinants may have a dual role in amine binding and/or maintenance of protein integrity. Importantly, a series of non-SRS elements are likely to be operative via long-range effects. While hydrophobic mechanisms appear to dominate orientation of the nitrogenous compounds toward the iron-oxene species, polar residues seem to foster binding events through H-bonding or salt-bridge formation. Careful uncovering of structure-function relationships in amine-enzyme association together with recently developed unsupervised machine learning approaches will be helpful in both tailoring of novel amine-type drugs and early elimination of potentially toxic or mutagenic candidates. Also, chimeragenesis might serve in the construction

  9. Perspective: On the active site model in computational catalyst screening

    NASA Astrophysics Data System (ADS)

    Reuter, Karsten; Plaisance, Craig P.; Oberhofer, Harald; Andersen, Mie

    2017-01-01

    First-principles screening approaches exploiting energy trends in surface adsorption represent an unparalleled success story in recent computational catalysis research. Here we argue that our still limited understanding of the structure of active sites is one of the major bottlenecks towards an ever extended and reliable use of such computational screening for catalyst discovery. For low-index transition metal surfaces, the prevalently chosen high-symmetry (terrace and step) sites offered by the nominal bulk-truncated crystal lattice might be justified. For more complex surfaces and composite catalyst materials, computational screening studies will need to actively embrace a considerable uncertainty with respect to what truly are the active sites. By systematically exploring the space of possible active site motifs, such studies might eventually contribute towards a targeted design of optimized sites in future catalysts.

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

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

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

  11. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report.

  12. Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

    PubMed

    Eaves, Deborah J; Haque, Tamanna; Tudor, Richard L; Barron, Yoshimi; Zampronio, Cleidiane G; Cotton, Nicholas P J; de Graaf, Barend H J; White, Scott A; Cooper, Helen J; Franklin, F Christopher H; Harper, Jeffery F; Franklin-Tong, Vernonica E

    2017-03-01

    Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca(2+) and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

  13. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  14. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

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

    1997-11-01

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

  15. Tritium activities in selected wells on the Nevada Test Site

    SciTech Connect

    Lyles, B.F.

    1993-05-01

    Literature and data were reviewed related to radionuclides in groundwater on and near the Nevada Test Site. No elevated tritium activities have been reported outside of the major testing regions of the Nevada Test Site. Three wells were identified as having water with above-background (>50 pCi/l) tritium activities: UE-15d Water Well; USGS Water Well A; and USGS Test Well B Ex. Although none of these wells have tritium activities greater than the Nevada State Drinking Water standard (20,000 pCi/l), their time-series tritium trends may be indicative to potential on-site radionuclide migration.

  16. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.

    PubMed

    Salsbury, Freddie R; Knutson, Stacy T; Poole, Leslie B; Fetrow, Jacquelyn S

    2008-02-01

    Cysteine sulfenic acid (Cys-SOH), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. This post-translational modification is not random: specific features near the cysteine control its reactivity. To identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known Cys-SOH sites) was compiled. Modifiable cysteines are found in proteins from most structural classes and many functional classes, but have no propensity for any one type of protein secondary structure. To identify features affecting cysteine reactivity, these sites were analyzed using both functional site profiling and electrostatic analysis. Overall, the solvent exposure of modifiable cysteines is not different from the average cysteine. The combined sequence, structure, and electrostatic approaches reveal mechanistic determinants not obvious from overall sequence comparison, including: (1) pKaS of some modifiable cysteines are affected by backbone features only; (2) charged residues are underrepresented in the structure near modifiable sites; (3) threonine and other polar residues can exert a large influence on the cysteine pKa; and (4) hydrogen bonding patterns are suggested to be important. This compilation of Cys-SOH modification sites and their features provides a quantitative assessment of previous observations and a basis for further analysis and prediction of these sites. Agreement with known experimental data indicates the utility of this combined approach for identifying mechanistic determinants at protein functional sites.

  17. Pattern similarity study of functional sites in protein sequences: lysozymes and cystatins

    PubMed Central

    Nakai, Shuryo; Li-Chan, Eunice CY; Dou, Jinglie

    2005-01-01

    Background Although it is generally agreed that topography is more conserved than sequences, proteins sharing the same fold can have different functions, while there are protein families with low sequence similarity. An alternative method for profile analysis of characteristic conserved positions of the motifs within the 3D structures may be needed for functional annotation of protein sequences. Using the approach of quantitative structure-activity relationships (QSAR), we have proposed a new algorithm for postulating functional mechanisms on the basis of pattern similarity and average of property values of side-chains in segments within sequences. This approach was used to search for functional sites of proteins belonging to the lysozyme and cystatin families. Results Hydrophobicity and β-turn propensity of reference segments with 3–7 residues were used for the homology similarity search (HSS) for active sites. Hydrogen bonding was used as the side-chain property for searching the binding sites of lysozymes. The profiles of similarity constants and average values of these parameters as functions of their positions in the sequences could identify both active and substrate binding sites of the lysozyme of Streptomyces coelicolor, which has been reported as a new fold enzyme (Cellosyl). The same approach was successfully applied to cystatins, especially for postulating the mechanisms of amyloidosis of human cystatin C as well as human lysozyme. Conclusion Pattern similarity and average index values of structure-related properties of side chains in short segments of three residues or longer were, for the first time, successfully applied for predicting functional sites in sequences. This new approach may be applicable to studying functional sites in un-annotated proteins, for which complete 3D structures are not yet available. PMID:15904486

  18. Proteome-wide analysis of nonsynonymous single-nucleotide variations in active sites of human proteins.

    PubMed

    Dingerdissen, Hayley; Motwani, Mona; Karagiannis, Konstantinos; Simonyan, Vahan; Mazumder, Raja

    2013-03-01

    An enzyme's active site is essential to normal protein activity such that any disruptions at this site may lead to dysfunction and disease. Nonsynonymous single-nucleotide variations (nsSNVs), which alter the amino acid sequence, are one type of disruption that can alter the active site. When this occurs, it is assumed that enzyme activity will vary because of the criticality of the site to normal protein function. We integrate nsSNV data and active site annotations from curated resources to identify all active-site-impacting nsSNVs in the human genome and search for all pathways observed to be associated with this data set to assess the likely consequences. We find that there are 934 unique nsSNVs that occur at the active sites of 559 proteins. Analysis of the nsSNV data shows an over-representation of arginine and an under-representation of cysteine, phenylalanine and tyrosine when comparing the list of nsSNV-impacted active site residues with the list of all possible proteomic active site residues, implying a potential bias for or against variation of these residues at the active site. Clustering analysis shows an abundance of hydrolases and transferases. Pathway and functional analysis shows several pathways over- or under-represented in the data set, with the most significantly affected pathways involved in carbohydrate metabolism. We provide a table of 32 variation-substrate/product pairs that can be used in targeted metabolomics experiments to assay the effects of specific variations. In addition, we report the significant prevalence of aspartic acid to histidine variation in eight proteins associated with nine diseases including glycogen storage diseases, lacrimo-auriculo-dento-digital syndrome, Parkinson's disease and several cancers.

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

    PubMed

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

    2016-06-17

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

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

    EPA Pesticide Factsheets

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

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

    PubMed

    Thune, I; Furberg, A S

    2001-06-01

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

  2. Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations.

    PubMed

    Schmitzer, Andreea R; Lépine, François; Pelletier, Joelle N

    2004-11-01

    We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a constant framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compositions. We determined that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The k(cat) and K(M) values were similar to those for the native enzyme. The favourable Delta(DeltaG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.

  3. 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

    2016-09-06

    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.

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

    PubMed Central

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

    2016-01-01

    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

  5. Site and Orbit Repeatabilities using Adaptive Mapping Functions

    NASA Astrophysics Data System (ADS)

    Desjardins, Camille; Gegout, Pascal; Soudarin, Laurent; Biancale, Richard; Perosanz, Felix

    2015-04-01

    The electromagnetic signals emitted by the satellite positioning systems travel at the speed of light in a straight line in a vacuum but are modified in their propagation through the neutral atmosphere by temporal and spatial changes of density, and composition and refractivity. These waves are slowed down and their trajectories are bent. This presentation summarizes the performances of the modeling of the tropospheric propagation by the ray tracing technique through the assimilations of the European Meteorological Centre (ECMWF) in the framework of realizing the geodetic reference frame. This goal is achieved by modeling the spatial variability of the propagation using the time variable three-dimensional physical parameters of the atmosphere. The tropospheric delays obtained by ray tracing in all directions throughout the meteorological model surrounding the geodetic site, are fitted by Adaptive Mapping Functions (AMF) parameterized by several tens of coefficients. The delays produced by the Horizon software are then experimented, kept unchanged or adjusted, when recovering a reference frame based on hundred sites using the GINS software. Without any adjustments of the tropospheric modeling, the subcentimetric performances of the AMF are demonstrated by the repeatability of sites positions and GPS satellites orbits. When some AMF coefficients are adjusted, the accuracy of orbits recovery in term of quadratic mean is 7 to 8 millimeters. This limit is imposed by the lack or deficiency of other models, such as non-tidal and tidal loading respectively. Hence the repeatability of the vertical position is not enhanced by changing the propagation model. At the contrary, the repeatability of the horizontal position of geodetic sites is greatly enhanced by accounting for the azimuthal variability provided by the realistic 3D shapes of the Atmosphere and the Earth and the rigorous interpolations of atmospheric parameters included in Adaptive Mapping Functions with respect

  6. Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori

    PubMed Central

    Damodaram, Kamala Jayanthi Pagadala; Kempraj, Vivek; Aurade, Ravindra Mahadappa; Rajasekhar, Sowmya Bandhisara; Venkataramanappa, Ravindra Kothapalli; Nandagopal, Bakthavatsalam; Verghese, Abraham

    2014-01-01

    Oviposition site-selection in insects is mediated through innate recognition templates (IRTs) tuned to specific chemical cues. These cues aid gravid insects in choosing suitable oviposition sites and may even enhance the fitness of their offspring by warding off predators and parasitoids. However, studies on the evolution of oviposition site-selection and cues instigating oviposition in domesticated insects remain elusive. Using the interaction between the silkmoth, Bombyx mori, and its host plant mulberry, Morus alba, as a model system, we demonstrate that centuries of domestication of silkmoth has not impaired its oviposition site-selection function. Silkmoths significantly preferred mulberry leaves to filter paper as oviposition sites. Oviposition assays with filter paper, filter paper treated with leaf volatiles and leaf alone proved that surface texture was not a significant criterion for oviposition site-selection, but volatile cues were. Oviposition assays with electrophysiologically active compounds from mulberry revealed that two of the volatiles, valencene and α-humulene, aided moths in choosing suitable oviposition sites and enhanced egg-laying significantly. Moreover, we show that generalist egg-parasitoids are strongly repelled by valencene and α-humulene. Our results demonstrate that IRTs tuned to cues that aid crucial functions like oviposition site-selection are less likely to be impaired even after centuries of domestication. PMID:25503440

  7. Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori.

    PubMed

    Damodaram, Kamala Jayanthi Pagadala; Kempraj, Vivek; Aurade, Ravindra Mahadappa; Rajasekhar, Sowmya Bandhisara; Venkataramanappa, Ravindra Kothapalli; Nandagopal, Bakthavatsalam; Verghese, Abraham

    2014-12-15

    Oviposition site-selection in insects is mediated through innate recognition templates (IRTs) tuned to specific chemical cues. These cues aid gravid insects in choosing suitable oviposition sites and may even enhance the fitness of their offspring by warding off predators and parasitoids. However, studies on the evolution of oviposition site-selection and cues instigating oviposition in domesticated insects remain elusive. Using the interaction between the silkmoth, Bombyx mori, and its host plant mulberry, Morus alba, as a model system, we demonstrate that centuries of domestication of silkmoth has not impaired its oviposition site-selection function. Silkmoths significantly preferred mulberry leaves to filter paper as oviposition sites. Oviposition assays with filter paper, filter paper treated with leaf volatiles and leaf alone proved that surface texture was not a significant criterion for oviposition site-selection, but volatile cues were. Oviposition assays with electrophysiologically active compounds from mulberry revealed that two of the volatiles, valencene and α-humulene, aided moths in choosing suitable oviposition sites and enhanced egg-laying significantly. Moreover, we show that generalist egg-parasitoids are strongly repelled by valencene and α-humulene. Our results demonstrate that IRTs tuned to cues that aid crucial functions like oviposition site-selection are less likely to be impaired even after centuries of domestication.

  8. A functional study of concanavalin A-histamine binding site overlap in Tetrahymena phagocytosis test.

    PubMed

    Csaba, G; Darvas, Z; László, V

    1983-01-01

    Treatment with histamine stimulated the phagocytotic activity of the Tetrahymena to a measurable degree, which was still demonstrable after a week (about 40 generations). Concanavalin A, which binds to the same membrane binding site as histamine, inhibited the stimulatory action of subsequently added histamine, but did not in itself influence phagocytotic activity in any way. The inhibitory effect of Con A on the histamine binding site proved to be dose-dependent. These observations stress the importance of investigating the functional context--as sole realistic measure--of receptor--ligand bindings.

  9. 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.

  10. Functional classification of protein 3D structures from predicted local interaction sites.

    PubMed

    Parasuram, Ramya; Lee, Joslynn S; Yin, Pengcheng; Somarowthu, Srinivas; Ondrechen, Mary Jo

    2010-12-01

    A new approach to the functional classification of protein 3D structures is described with application to some examples from structural genomics. This approach is based on functional site prediction with THEMATICS and POOL. THEMATICS employs calculated electrostatic potentials of the query structure. POOL is a machine learning method that utilizes THEMATICS features and has been shown to predict accurate, precise, highly localized interaction sites. Extension to the functional classification of structural genomics proteins is now described. Predicted functionally important residues are structurally aligned with those of proteins with previously characterized biochemical functions. A 3D structure match at the predicted local functional site then serves as a more reliable predictor of biochemical function than an overall structure match. Annotation is confirmed for a structural genomics protein with the ribulose phosphate binding barrel (RPBB) fold. A putative glucoamylase from Bacteroides fragilis (PDB ID 3eu8) is shown to be in fact probably not a glucoamylase. Finally a structural genomics protein from Streptomyces coelicolor annotated as an enoyl-CoA hydratase (PDB ID 3g64) is shown to be misannotated. Its predicted active site does not match the well-characterized enoyl-CoA hydratases of similar structure but rather bears closer resemblance to those of a dehalogenase with similar fold.

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

    PubMed Central

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

    2015-01-01

    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 (Mw 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

  12. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

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

  13. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism.

    PubMed

    Medina-Carmona, Encarnación; Neira, Jose L; Salido, Eduardo; Fuchs, Julian E; Palomino-Morales, Rogelio; Timson, David J; Pey, Angel L

    2017-03-14

    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

  14. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

    NASA Astrophysics Data System (ADS)

    Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

    2017-03-01

    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

  15. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

    PubMed Central

    Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

    2017-01-01

    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention. PMID:28291250

  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. 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.

  18. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

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

    PubMed Central

    2016-01-01

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

  20. Incorporating distant sequence features and radial basis function networks to identify ubiquitin conjugation sites.

    PubMed

    Lee, Tzong-Yi; Chen, Shu-An; Hung, Hsin-Yi; Ou, Yu-Yen

    2011-03-09

    Ubiquitin (Ub) is a small protein that consists of 76 amino acids about 8.5 kDa. In ubiquitin conjugation, the ubiquitin is majorly conjugated on the lysine residue of protein by Ub-ligating (E3) enzymes. Three major enzymes participate in ubiquitin conjugation. They are E1, E2 and E3 which are responsible for activating, conjugating and ligating ubiquitin, respectively. Ubiquitin conjugation in eukaryotes is an important mechanism of the proteasome-mediated degradation of a protein and regulating the activity of transcription factors. Motivated by the importance of ubiquitin conjugation in biological processes, this investigation develops a method, UbSite, which uses utilizes an efficient radial basis function (RBF) network to identify protein ubiquitin conjugation (ubiquitylation) sites. This work not only investigates the amino acid composition but also the structural characteristics, physicochemical properties, and evolutionary information of amino acids around ubiquitylation (Ub) sites. With reference to the pathway of ubiquitin conjugation, the substrate sites for E3 recognition, which are distant from ubiquitylation sites, are investigated. The measurement of F-score in a large window size (-20∼+20) revealed a statistically significant amino acid composition and position-specific scoring matrix (evolutionary information), which are mainly located distant from Ub sites. The distant information can be used effectively to differentiate Ub sites from non-Ub sites. As determined by five-fold cross-validation, the model that was trained using the combination of amino acid composition and evolutionary information performs best in identifying ubiquitin conjugation sites. The prediction sensitivity, specificity, and accuracy are 65.5%, 74.8%, and 74.5%, respectively. Although the amino acid sequences around the ubiquitin conjugation sites do not contain conserved motifs, the cross-validation result indicates that the integration of distant sequence features of Ub

  1. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

    PubMed Central

    Breunig, K D; Kuger, P

    1987-01-01

    As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription. Images PMID:2830492

  2. In vivo suppressive function of myeloid-derived suppressor cells is limited to the inflammatory site

    PubMed Central

    Haverkamp, Jessica M.; Crist, Scott A.; Elzey, Bennett D.; Cimen, Cansu; Ratliff, Timothy L.

    2011-01-01

    Current thinking suggests that despite the heterogeneity of myeloid-derived suppressor cells (MDSC), all Gr-1+CD11b+ cells can become suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSC function enhances T cell responses. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. We now demonstrate that during a tissue specific inflammatory response, MDSC inhibition of CD8 T cell proliferation and IFN-γ production is restricted to the inflammatory site. Using a prostate specific inflammatory model and a heterotopic prostate tumor model, we show that MDSC from inflammatory sites or from tumor tissue possess immediate capacity to inhibit T cell function, whereas those isolated from peripheral tissues (spleens and liver) are not suppressive without activation of iNOS by exposure to IFN-γ. These data show MDSC are important regulators of immune responses in the prostate during acute inflammation and the chronic inflammatory setting of tumor growth and that regulation of T cell function by MDSC during a localized inflammatory response is restricted in vivo to the site of an ongoing immune response. PMID:21287554

  3. 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

  4. Functional Relationships between Agonist Binding Sites and Coupling Regions of Homomeric Cys-Loop Receptors

    PubMed Central

    Andersen, Natalia; Corradi, Jeremías; Bartos, Mariana

    2011-01-01

    Each subunit in a homopentameric Cys-loop receptor contains a specialized coupling region positioned between the agonist binding domain and the ion conductive channel. To determine the contribution of each coupling region to the stability of the open channel, we constructed a receptor subunit (α7-5-HT3A) with both a disabled coupling region and a reporter mutation that alters unitary conductance, and coexpressed normal and mutant subunits. The resulting receptors show single-channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of the number of intact coupling regions with mean open time. We find that each coupling region contributes an equal increment to the stability of the open channel. However, by altering the numbers and locations of active coupling regions and binding sites, we find that a coupling region in a subunit flanked by inactive binding sites can still stabilize the open channel. We also determine minimal requirements for channel opening regardless of stability and find that channel opening can occur in a receptor with one active coupling region flanked by functional binding sites or with one active binding site flanked by functional coupling regions. The overall findings show that, whereas the agonist binding sites contribute interdependently and asymmetrically to open-channel stability, the coupling regions contribute independently and symmetrically. PMID:21389221

  5. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.

    PubMed

    Dian, Cyril; Vitale, Sylvia; Leonard, Gordon A; Bahlawane, Christelle; Fauquant, Caroline; Leduc, Damien; Muller, Cécile; de Reuse, Hilde; Michaud-Soret, Isabelle; Terradot, Laurent

    2011-03-01

    Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS₄ site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

  6. Prediction of biological functions on glycosylation site migrations in human influenza H1N1 viruses.

    PubMed

    Sun, Shisheng; Wang, Qinzhe; Zhao, Fei; Chen, Wentian; Li, Zheng

    2012-01-01

    Protein glycosylation alteration is typically employed by various viruses for escaping immune pressures from their hosts. Our previous work had shown that not only the increase of glycosylation sites (glycosites) numbers, but also glycosite migration might be involved in the evolution of human seasonal influenza H1N1 viruses. More importantly, glycosite migration was likely a more effectively alteration way for the host adaption of human influenza H1N1 viruses. In this study, we provided more bioinformatics and statistic evidences for further predicting the significant biological functions of glycosite migration in the host adaptation of human influenza H1N1 viruses, by employing homology modeling and in silico protein glycosylation of representative HA and NA proteins as well as amino acid variability analysis at antigenic sites of HA and NA. The results showed that glycosite migrations in human influenza viruses have at least five possible functions: to more effectively mask the antigenic sites, to more effectively protect the enzymatic cleavage sites of neuraminidase (NA), to stabilize the polymeric structures, to regulate the receptor binding and catalytic activities and to balance the binding activity of hemagglutinin (HA) with the release activity of NA. The information here can provide some constructive suggestions for the function research related to protein glycosylation of influenza viruses, although these predictions still need to be supported by experimental data.

  7. Functionalized active-nucleus complex sensor

    DOEpatents

    Pines, Alexander; Wemmer, David E.; Spence, Megan; Rubin, Seth

    2003-11-25

    A functionalized active-nucleus complex sensor that selectively associates with one or more target species, and a method for assaying and screening for one or a plurality of target species utilizing one or a plurality of functionalized active-nucleus complexes with at least two of the functionalized active-nucleus complexes having an attraction affinity to different corresponding target species. The functionalized active-nucleus complex has an active-nucleus and a targeting carrier. The method involves functionalizing an active-nucleus, for each functionalized active-nucleus complex, by incorporating the active-nucleus into a macromolucular or molecular complex that is capable of binding one of the target species and then bringing the macromolecular or molecular complexes into contact with the target species and detecting the occurrence of or change in a nuclear magnetic resonance signal from each of the active-nuclei in each of the functionalized active-nucleus complexes.

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

    PubMed

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

    2017-01-16

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Biochemical analysis of active site mutations of human polymerase η.

    PubMed

    Suarez, Samuel C; Beardslee, Renee A; Toffton, Shannon M; McCulloch, Scott D

    2013-01-01

    DNA polymerase η (pol η) plays a critical role in suppressing mutations caused by the bypass of cis-syn cyclobutane pyrimidine dimers (CPD) that escape repair. There is evidence this is also the case for the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG). Both of these lesions cause moderate to severe blockage of synthesis when encountered by replicative polymerases, while pol η displays little no to pausing during translesion synthesis. However, since lesion bypass does not remove damaged DNA from the genome and can possibly be accompanied by errors in synthesis during bypass, the process is often called 'damage tolerance' to delineate it from classical DNA repair pathways. The fidelity of lesion bypass is therefore of importance when determining how pol η suppresses mutations after DNA damage. As pol η has been implicated in numerous in vivo pathways other than lesion bypass, we wanted to better understand the molecular mechanisms involved in the relatively low-fidelity synthesis displayed by pol η. To that end, we have created a set of mutant pol η proteins each containing a single amino acid substitution in the active site and closely surrounding regions. We determined overall DNA synthesis ability as well as the efficiency and fidelity of bypass of thymine-thymine CPD (T-T CPD) and 8-oxoG containing DNA templates. Our results show that several amino acids are critical for normal polymerase function, with changes in overall activity and fidelity being observed. Of the mutants that retain polymerase activity, we demonstrate that amino acids Q38, Y52, and R61 play key roles in determining polymerase fidelity, with substation of alanine causing both increases and decreases in fidelity. Remarkably, the Q38A mutant displays increased fidelity during synthesis opposite 8-oxoG but decreased fidelity during synthesis opposite a T-T CPD. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity

    PubMed Central

    Chappell, Stephen A.; Edelman, Gerald M.; Mauro, Vincent P.

    2000-01-01

    This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′ untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′ UTR that is 100% complementary to the 18S rRNA at nucleotides 1132–1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements. PMID:10677496

  11. 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.

  12. Discriminative structural approaches for enzyme active-site prediction

    PubMed Central

    2011-01-01

    Background Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. Results This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. Conclusions This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses. PMID:21342581

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

    PubMed

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

    2010-12-01

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

  14. Identification of thromboxane A2 synthase active site residues by molecular modeling-guided site-directed mutagenesis.

    PubMed

    Wang, L H; Matijevic-Aleksic, N; Hsu, P Y; Ruan, K H; Wu, K K; Kulmacz, R J

    1996-08-16

    Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structure-function relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting

  15. 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.

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

    SciTech Connect

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

    2015-03-25

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

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

    DOE PAGES

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

    2015-03-25

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

  18. Active site and remote contributions to catalysis in methylthioadenosine nucleosidases.

    PubMed

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

    2015-04-21

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

  19. Site-directed mutagenesis of IRX9, IRX9L and IRX14 proteins involved in xylan biosynthesis: glycosyltransferase activity is not required for IRX9 function in Arabidopsis.

    PubMed

    Ren, Yanfang; Hansen, Sara Fasmer; Ebert, Berit; Lau, Jane; Scheller, Henrik Vibe

    2014-01-01

    Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic

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

    PubMed

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

    2015-03-04

    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.

  1. Site-specific methylated reporter constructs for functional analysis of DNA methylation.

    PubMed

    Han, Weiguo; Shi, Miao; Spivack, Simon D

    2013-11-01

    Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p<0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p<0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (> 8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

  2. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  3. 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.

  4. A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence

    PubMed Central

    Liang, Wen-Wei; Cheng, Soo-Chen

    2015-01-01

    The DEAD-box RNA helicase Prp5 is required for the formation of the prespliceosome through an ATP-dependent function to remodel U2 small nuclear ribonucleoprotein particles (snRNPs) and an ATP-independent function of unknown mechanism. Prp5 has also been implicated in proofreading the branch site sequence, but the molecular mechanism has not been well characterized. Using actin precursor mRNA (pre-mRNA) carrying branch site mutations, we identified a Prp5-containing prespliceosome with Prp5 directly bound to U2 small nuclear RNA (snRNA). Prp5 is in contact with U2 in regions on and near the branchpoint-interacting stem–loop (BSL), suggesting that Prp5 may function in stabilizing the BSL. Regardless of its ATPase activity, Prp5 mutants that suppress branch site mutations associate with the spliceosome less tightly and allow more tri-snRNP binding for the reaction to proceed. Our results suggest a novel mechanism for how Prp5 functions in prespliceosome formation and proofreading of the branch site sequence. Prp5 binds to the spliceosome in association with U2 by interacting with the BSL and is released upon the base-pairing of U2 with the branch site to allow the recruitment of the tri-snRNP. Mutations impairing U2–branch site base-pairing retard Prp5 release and impede tri-snRNP association. Prp5 mutations that destabilize the Prp5–U2 interaction suppress branch site mutations by allowing progression of the pathway. PMID:25561497

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

    DOE PAGES

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

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.« less

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

    SciTech Connect

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; Nørskov, Jens K.; Studt, Felix

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidation reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.

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

    SciTech Connect

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; Nørskov, Jens K.; Studt, Felix

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidation reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.

  8. Dendritic Encapsulation of Function: Applying Nature's Site Isolation Principle from Biomimetics to Materials Science.

    PubMed

    Hecht, Stefan; Fréchet, Jean M. J.

    2001-01-05

    The convergence of our understanding of structure-property relationships for selected biological macromolecules and our increased ability to prepare large synthetic macromolecules with a structural precision that approaches that of proteins have spawned a new area of research where chemistry and materials science join with biology. While evolution has enabled nature to perfect processes involving energy transfer or catalysis by incorporating functions such as self-replication and repair, synthetic macromolecules still depend on our synthetic skills and abilities to mesh structure and function in our designs. Clearly, we can take advantage of our understanding of natural systems to mimic the structural features that lead to optimized function. For example, numerous biological systems make use of the concept of site isolation whereby an active center or catalytic site is encapsulated, frequently within a protein, to afford properties that would not be encountered in the bulk state. The ability of the dendritic shell to encapsulate functional core moieties and to create specific site-isolated nanoenvironments, and thereby affect molecular properties, has been explored. By utilizing the distinct properties of the dendrimer architecture active sites that have either photophysical, photochemical, electrochemical, or catalytic functions have been placed at the core. Applying the general concept of site isolation to problems in materials research is likely to prove extremely fruitful in the long term, with short-term applications in areas such as the construction of improved optoelectronic devices. This review focuses on the evolution of a natural design principle that contributes to bridging the gap between biology and materials science. The recent progress in the synthesis of dendrimer-encapsulated molecules and their study by a variety of techniques is discussed. These investigations have implications that range from the preliminary design of artificial enzymes

  9. 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…

  10. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  11. 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…

  12. 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.

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

    SciTech Connect

    Carra,J.; McHugh, C.; Mulligan, S.; Machiesky, L.; Soares, A.; Millard, C.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

  14. Direct instrumental identification of catalytically active surface sites

    NASA Astrophysics Data System (ADS)

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

    2017-09-01

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

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

    SciTech Connect

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

    1994-04-01

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

  16. 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

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

    PubMed

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

    2007-06-26

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

  19. Functional arginine residue in the catalytic site of the tonoplast H/sup +/-ATPase

    SciTech Connect

    Bennett, A.B.; Borcherts, K.

    1987-04-01

    Phenylglyoxal was used to convalently modify arginine residues of the red beet tonoplast H/sup +/-ATPase. Phenylglyoxal completely inactivated ATPase activity. Inactivation followed pseudo-first-order kinetics with an apparent reaction order near one, suggesting that enzyme inactivation resulted from modification of a single arginine residue per enzyme molecule. Potassium salts of several anions partially protected for phenylglyoxal inactivation by this appeared to be non-specific since anions that stimulate H/sup +/-ATPase activity were not the most effective in protecting activity. Similarly, 100 um DIDS was only slightly effective in protecting from phenylglyoxal inhibition. ATP protected from phenylglyoxal inactivation while ADP did not and phenylglyoxal inhibition decreased the Vmax without effecting the Km for ATP hydrolysis. These results suggest that functional arginine modification occurs at or near the catalytic ATP binding site. Attempts to label this site with (/sup 14/C)phenylglyoxal will be presented.

  20. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

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

    PubMed

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

    2003-05-13

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

  2. 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.

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

    SciTech Connect

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

    2009-11-10

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

  4. Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen

    PubMed Central

    Phillips, John D.; Warby, Christy A.; Whitby, Frank G.; Kushner, James P.; Hill, Christopher P.

    2009-01-01

    Summary 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 the four acetate side chains on 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 (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the 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 create a large active site cleft. PMID:19362562

  5. Transcriptional activation by LR1 at the Eµ enhancer and switch region sites

    PubMed Central

    Hanakahi, L. A.; Maizels, Nancy

    2000-01-01

    LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Eµ immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Eµ core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Eµ core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions. PMID:10908319

  6. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  7. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    PubMed Central

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-01-01

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin. PMID:3355517

  8. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    PubMed

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-02-15

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

  9. Crystal structure of plant ferritin reveals a novel metal binding site that functions as a transit site for metal transfer in ferritin.

    PubMed

    Masuda, Taro; Goto, Fumiyuki; Yoshihara, Toshihiro; Mikami, Bunzo

    2010-02-05

    Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-A resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu(173) and carbonyl oxygen of Thr(168), which correspond, respectively, to Glu(140) and Thr(135) of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

  10. 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.

  11. Identification of functional regions in the Rhodospirillum rubrum L-asparaginase by site-directed mutagenesis.

    PubMed

    Pokrovskaya, M V; Aleksandrova, S S; Pokrovsky, V S; Veselovsky, A V; Grishin, D V; Abakumova, O Yu; Podobed, O V; Mishin, A A; Zhdanov, D D; Sokolov, N N

    2015-03-01

    Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

  12. 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.

  13. Distribution bias analysis of germline and somatic single-nucleotide variations that impact protein functional site and neighboring amino acids

    PubMed Central

    Pan, Yang; Yan, Cheng; Hu, Yu; Fan, Yu; Pan, Qing; Wan, Quan; Torcivia-Rodriguez, John; Mazumder, Raja

    2017-01-01

    Single nucleotide variations (SNVs) can result in loss or gain of protein functional sites. We analyzed the effects of SNVs on enzyme active sites, ligand binding sites, and various types of post translational modification (PTM) sites. We found that, for most types of protein functional sites, the SNV pattern differs between germline and somatic mutations as well as between synonymous and non-synonymous mutations. From a total of 51,138 protein functional site affecting SNVs (pfsSNVs), a pan-cancer analysis revealed 142 somatic pfsSNVs in five or more cancer types. By leveraging patient information for somatic pfsSNVs, we identified 17 loss of functional site SNVs and 60 gain of functional site SNVs which are significantly enriched in patients with specific cancer types. Of the key pfsSNVs identified in our analysis above, we highlight 132 key pfsSNVs within 17 genes that are found in well-established cancer associated gene lists. For illustrating how key pfsSNVs can be prioritized further, we provide a use case where we performed survival analysis showing that a loss of phosphorylation site pfsSNV at position 105 in MEF2A is significantly associated with decreased pancreatic cancer patient survival rate. These 132 pfsSNVs can be used in developing genetic testing pipelines. PMID:28176830

  14. Biochemical characterization of the chondroitinase ABC I active site

    PubMed Central

    2005-01-01

    cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via β-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme–substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme–substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis. PMID:16108757

  15. Biochemical characterization of the chondroitinase ABC I active site.

    PubMed

    Prabhakar, Vikas; Raman, Rahul; Capila, Ishan; Bosques, Carlos J; Pojasek, Kevin; Sasisekharan, Ram

    2005-09-01

    cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via beta-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme-substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme-substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.

  16. Neuroprotection by a novel NMDAR functional glycine site partial agonist, GLYX-13.

    PubMed

    Stanton, Patric K; Potter, Pamela E; Aguilar, Jennifer; Decandia, Maria; Moskal, Joseph R

    2009-08-26

    GLYX-13 (threonine-proline-proline-threonine-amide) is an amidated di-pyrrolidine that acts as a functional partial agonist at the glycine site on N-methyl-D-aspartate glutamate receptors (NMDARs). GLYX-13 can both increase NMDAR conductance at NR2B-containing receptors, and reduce conductance of non-NR2B-containing receptors. Here, we report that GLYX-13 potently reduces delayed (24 h) death of CA1 pyramidal neurons produced by bilateral carotid occlusion in Mongolian gerbils, when administered up to 5 h post-ischemia. GLYX-13 also reduced delayed (24 h) neuronal death of CA1, CA3, and dentate gyrus principal neurons elicited by oxygen/glucose deprivation in in-vitro hippocampal organotypic slice cultures, when applied up to 2 h post-oxygen/glucose deprivation. The glycine site full agonist D-serine completely occluded neuroprotection, indicating that GLYX-13 acts by modulating activation of this site.

  17. Ground Truth and Application for the Anisotropic Receiver Functions Technique - Test site KTB: the installation campaign

    NASA Astrophysics Data System (ADS)

    Bianchi, Irene; Anselmi, Mario; Apoloner, Maria-Theresia; Qorbani, Ehsan; Gribovszki, Katalin; Bokelmann, Götz

    2015-04-01

    The project at hand is a field test around the KTB (Kontinentale Tiefbohrung) site in the Oberpfalz, Southeastern Germany, at the northwestern edge of the Bohemian Massif. The region has been extensively studied through the analysis of several seismic reflection lines deployed around the drilling site. The deep borehole had been placed into gneiss rocks of the Zone Erbendorf-Vohenstrauss. Drilling activity lasted since 1987 to 1994, and it descends down to a depth of 9101 meters. In our experiment, we aim to recover structural information as well as anisotropy of the upper crust using the receiver function technique. This retrieved information will form the base for a comparison between the resulting anisotropy amount and orientation with information of rock samples from up to 9 km depth, and with earlier high-frequency seismic experiments around the drill site. For that purpose, we installed 9 seismic stations, and recorded seismicity continuously for two years.

  18. Active mutants of the TCR-mediated p38α alternative activation site show changes in the phosphorylation lip and DEF site formation.

    PubMed

    Tzarum, Netanel; Diskin, Ron; Engelberg, David; Livnah, Oded

    2011-02-04

    The p38α mitogen-activated protein kinase is commonly activated by dual (Thr and Tyr) phosphorylation catalyzed by mitogen-activated protein kinase kinases. However, in T-cells, upon stimulation of the T-cell receptor, p38α is activated via an alternative pathway, involving its phosphorylation by zeta-chain-associated protein kinase 70 on Tyr323, distal from the phosphorylation lip. Tyr323-phosphorylated p38α is autoactivated, resulting in monophosphorylation of Thr180. The conformational changes induced by pTyr323 mediating autoactivation are not known. The lack of pTyr323 p38α for structural studies promoted the search for Tyr323 mutations that may functionally emulate its effect when phosphorylated. Via a comprehensive mutagenesis of Tyr323, we identified mutations that rendered the kinase intrinsically active and others that displayed no activity. Crystallographic studies of selected active (p38α(Y323Q), p38α(Y323T), and p38α(Y323R)) and inactive (p38α(Y323F)) mutants revealed that substantial changes in interlobe orientation, extended conformation of the activation loop, and formation of substrate docking DEF site (docking site for extracellular signal-regulated kinase FXF) interaction pocket are associated with p38α activation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. An Active MammoSite for Breast Cancer Treatment

    NASA Astrophysics Data System (ADS)

    Quan, Alice

    2007-03-01

    Breast brachytherapy using the MammoSite balloon catheter is one of the latest developments in breast cancer treatment and is the most performed method of brachytherapy. A high activity ^192Ir radioactive source is pushed inside the shaft of the device until it reaches the center of the balloon. The latest involvement of the Brachytherapy research group of the medical physics program at Hampton University is in the development of a scintillating fiber based detector for the breast cancer specific MammoSite balloon catheter from Cytyc, Inc. During the summer 2006, data were acquired at a local hospital (Bon Secours DePaul Medical Center) to evaluate the possibility of measuring the source location and dose distribution during breast brachytherapy cancer treatments with this device. Two 0.5 mm^2 and 1.0 mm^2 scintillating fibers were used for these experiments. We used two modified MammoSite devices, each housing an extra tubing within which the fibers were inserted. The results from these runs confirm the possibility of an active MammoSite to monitor the location of the source as well its dose distribution during patient treatment. We will describe the experimental setup and discuss the data.

  20. Functional integrity of the interrenal tissue of yellow perch from contaminated sites tested in vivo

    SciTech Connect

    Girard, C.; Brodeur, J.C.; Hontela, A.

    1995-12-31

    The normal activation of the hypothalamo-pituitary-interrenal axis (HPI axis) in response to capture is disrupted in fish subjected to life-long exposure to heavy metals, PCBs and PAHs. The ability to increase plasma cortisol in yellow perch (Perca flavescens) from sites contaminated by heavy metals and organic compounds, and from a reference site was assessed by the Capture stress test and by the ACTH Challenge test, a new standardized in vivo method designed for field studies. The effects of seasonal factors, such as temperature and gonadal maturity on these tests were investigated. Measures of liver and muscle glycogen and histopathology were made to further characterize the biochemical and structural changes that may occur along with hormonal changes. The Capture stress test showed that an acute source of stress induced a lower cortisol response in fish from the highly contaminated site compared to the reference site, revealing a functional impairment of the HPI axis. The ACTH Challenge test showed that the hormonal responsiveness of the cortisol-secreting interrenal tissue, stimulated by a standard dose of ACTH injected i.p., was lower in fish from the highly contaminated site than the reference site. Spring is the season during which the impairment was the most evident. The possibility of using the reduced capacity of feral fish to respond to a standardized ACTH Challenge as an early bioindicator of toxic stress is discussed.

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  2. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  3. Communication: Active space decomposition with multiple sites: density matrix renormalization group algorithm.

    PubMed

    Parker, Shane M; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE(h) or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  4. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

    Edges are special sites in nanomaterials. The atoms residing on the edges have different environments compared to those in other parts of a nanomaterial and, therefore, they may have different properties. Here, recent progress in nanomaterial fields is summarized from the viewpoint of the edges. Typically, edge sites in MoS2 or metals, other than surface atoms, can perform as active centers for catalytic reactions, so the method to enhance performance lies in the optimization of the edge structures. The edges of multicomponent interfaces present even more possibilities to enhance the activities of nanomaterials. Nanoframes and ultrathin nanowires have similarities to conventional edges of nanoparticles, the application of which as catalysts can help to reduce the use of costly materials. Looking beyond this, the edge structures of graphene are also essential for their properties. In short, the edge structure can influence many properties of materials. PMID:27980960

  5. Modification of the binding site(s) of lectins by an affinity column carrying an activated galactose-terminated ligand.

    PubMed

    Moroney, S E; D'Alarcao, L J; Goldmacher, V S; Lambert, J M; Blättler, W A

    1987-12-15

    An affinity column approach is described, aimed at the modification of the galactose binding site(s) of ricin in an effort to block the binding of ricin to cells. The affinity column was prepared by linking N-(2'-mercaptoethyl)lactamine to pyridyldithio-activated polyacrylamide heads. The linker between the ligand and the solid support thus contained a disulfide bond and an unmodified terminal galactose moiety. The amino group of the ligand was allowed to react with the bifunctional cross-linking reagent 2,4-dichloro-6-methoxytriazine. The lectin was then allowed to bind to the galactose functions on the activated column at pH 7.0, prior to raising the pH to 8.6 to initiate the cross-linking reaction between the ligand and the lectin. Lectin that was not covalently linked to the functionalized galactose residues on the column was eluted with galactose or lactose. Finally, the covalent ligand-lectin complexes were released from the solid support by reducing the disulfide bond between the ligand and the support. The affinity column was used in this way to modify the galactose binding site(s) of ricin. Upon release from the affinity column, blocked ricin was purified from unmodified ricin by affinity chromatography on columns of immobilized asialofetuin (a ligand to which ricin binds very tightly). The sulfhydryl group formed by cleavage of the ligand-ricin complex from the column was labeled with [3H]-N-ethylmaleimide to provide evidence that one blocking ligand was linked per ricin molecule. The blocked ricin and a conjugate of the blocked ricin with the monoclonal antibody J5 were toxic for cultures of Namalwa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

  7. The inhibition of proinsulin-processing endopeptidase activities by active-site-directed peptides.

    PubMed Central

    Rhodes, C J; Zumbrunn, A; Bailyes, E M; Shaw, E; Hutton, J C

    1989-01-01

    Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities. PMID:2649090

  8. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  9. 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.

  10. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  11. Di/mono-nuclear iron(I)/(II) complexes as functional models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases: protonations, molecular structures and electrochemical properties.

    PubMed

    Gao, Shang; Fan, Jiangli; Sun, Shiguo; Song, Fengling; Peng, Xiaojun; Duan, Qian; Jiang, Dayong; Liang, Qingcheng

    2012-10-21

    Di/mono-nuclear iron(I)/(II) complexes containing conjugated and electron-withdrawing S-to-S linkers, [{(μ-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(6)] (1), [{(μ-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(5)(PMe(3))] (1P), and [{(μ-S)(2)(C(4)N(2)H(2))}Fe(CO)(2)(PMe(3))(2)] (2) were prepared as biomimetic models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases. The N atoms in the heterocyclic pyrazines of 1 and 2 were protonated in the presence of proton acid to generate one and two hydrides, [1(NH)](+) CF(3)SO(3)(-), [2(NH)](+) CF(3)SO(3)(-), and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2), respectively. The protonation processes were evidenced by in situ IR and NMR spectroscopy. The molecular structures of the protonated species [1(NH)](+) CF(3)SO(3)(-) and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2) together with their originating complexes and , and the mono-PMe(3) substituted diiron complex were identified by X-ray crystallography. The IR and single-crystal analysis data all suggested that the electron-withdrawing bridge, pyrazine, led to decreased electron density at the Fe centers of the model complexes, which was consistent with the electrochemical studies. The cyclic voltammograms indicated that complex exhibited a low primary reduction potential at -1.17 V vs. Fc-Fc(+) with a 270 mV positive shift compared with that of the benzene-1,2-dithiolate (bdt) bridged analogue [(μ-bdt)Fe(2)(CO)(6)]. Under the weak acid conditions, complexes 1 and 2 could electrochemically catalyze the proton reduction. More interestingly, the mononuclear ferrous complex 2 showed two catalytic peaks during the formation of hydrogen, confirming its potential as a catalyst for hydrogen production.

  12. Dual function of a nuclear factor I binding site in MMTV transcription regulation.

    PubMed Central

    Buetti, E; Kühnel, B; Diggelmann, H

    1989-01-01

    Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element. Images PMID:2542892

  13. Interactive flare sites within an active region complex

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2016-11-01

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

  17. Are Synonymous Sites in Primates and Rodents Functionally Constrained?

    PubMed

    Price, Nicholas; Graur, Dan

    2016-01-01

    It has been claimed that synonymous sites in mammals are under selective constraint. Furthermore, in many studies the selective constraint at such sites in primates was claimed to be more stringent than that in rodents. Given the larger effective population sizes in rodents than in primates, the theoretical expectation is that selection in rodents would be more effective than that in primates. To resolve this contradiction between expectations and observations, we used processed pseudogenes as a model for strict neutral evolution, and estimated selective constraint on synonymous sites using the rate of substitution at pseudosynonymous and pseudononsynonymous sites in pseudogenes as the neutral expectation. After controlling for the effects of GC content, our results were similar to those from previous studies, i.e., synonymous sites in primates exhibited evidence for higher selective constraint that those in rodents. Specifically, our results indicated that in primates up to 24% of synonymous sites could be under purifying selection, while in rodents synonymous sites evolved neutrally. To further control for shifts in GC content, we estimated selective constraint at fourfold degenerate sites using a maximum parsimony approach. This allowed us to estimate selective constraint using mutational patterns that cause a shift in GC content (GT ↔ TG, CT ↔ TC, GA ↔ AG, and CA ↔ AC) and ones that do not (AT ↔ TA and CG ↔ GC). Using this approach, we found that synonymous sites evolve neutrally in both primates and rodents. Apparent deviations from neutrality were caused by a higher rate of C → A and C → T mutations in pseudogenes. Such differences are most likely caused by the shift in GC content experienced by pseudogenes. We conclude that previous estimates according to which 20-40% of synonymous sites in primates were under selective constraint were most likely artifacts of the biased pattern of mutation.

  18. Multimorbidity, cognitive function, and physical activity.

    PubMed

    Loprinzi, Paul D

    2016-02-01

    Previous research demonstrates that both physical activity and multimorbidity are associated with cognitive function. However, the extent to which physical activity may moderate the relationship between multimorbidity and cognitive function has not been thoroughly evaluated. Data from the 1999-2002 NHANES were used (60+ years; N = 2157). A multimorbidity index variable was created based on physician diagnosis of a multitude of chronic diseases. Physical activity was self-reported and cognitive function was evaluated from the digit symbol substitution test. Multimorbidity was inversely associated with cognitive function for the unadjusted and adjusted models. However, generally, multimorbidity was no longer associated with cognitive function for the majority of older adults who achieved the minimum recommended physical activity level (≥2000 MET-min-month), as issued by the United States Department of Health and Human Services. In this national sample of older adults, there was some evidence to suggest that physical activity moderates the relationship between multimorbidity and cognitive function.

  19. Control of the active site structure of giant bilayer hemoglobin from the Annelid Eisenia foetida using hierarchic assemblies

    SciTech Connect

    Girasole, Marco; Arcovito, Alessandro; Marconi, Augusta; Davoli, Camilla; Congiu-Castellano, Agostina; Bellelli, Andrea; Amiconi, Gino

    2005-12-05

    The active site structure of the oxygenated derivative of the main subassemblies (whole protein, dodecamers, and trimers) of the giant haemoglobin from Eisenia foetida has been characterized by x-ray absorption near edge structure spectroscopy. The data revealed a remarkable effect of the hierarchic assemblies on the active site of the subunit. Specifically, the whole protein has the same site structure of the dodecamer, while a sharp conformational transition occurs when the dodecamer is disassembled into trimers (and monomers) revealing that constraints due to the protein matrix determine the active site geometry and, consequently, the protein function in these large complexes.

  20. Control of the active site structure of giant bilayer hemoglobin from the Annelid Eisenia foetida using hierarchic assemblies

    NASA Astrophysics Data System (ADS)

    Girasole, Marco; Arcovito, Alessandro; Marconi, Augusta; Davoli, Camilla; Congiu-Castellano, Agostina; Bellelli, Andrea; Amiconi, Gino

    2005-12-01

    The active site structure of the oxygenated derivative of the main subassemblies (whole protein, dodecamers, and trimers) of the giant haemoglobin from Eisenia foetida has been characterized by x-ray absorption near edge structure spectroscopy. The data revealed a remarkable effect of the hierarchic assemblies on the active site of the subunit. Specifically, the whole protein has the same site structure of the dodecamer, while a sharp conformational transition occurs when the dodecamer is disassembled into trimers (and monomers) revealing that constraints due to the protein matrix determine the active site geometry and, consequently, the protein function in these large complexes.

  1. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

  2. Geo-Chip analysis reveals reduced functional diversity of the bacterial community at a dumping site for dredged Elbe sediment.

    PubMed

    Störmer, Rebecca; Wichels, Antje; Gerdts, Gunnar

    2013-12-15

    The dumping of dredged sediments represents a major stressor for coastal ecosystems. The impact on the ecosystem function is determined by its complexity not easy to assess. In the present study, we evaluated the potential of bacterial community analyses to act as ecological indicators in environmental monitoring programmes. We investigated the functional structure of bacterial communities, applying functional gene arrays (GeoChip4.2). The relationship between functional genes and environmental factors was analysed using distance-based multivariate multiple regression. Apparently, both the function and structure of the bacterial communities are impacted by dumping activities. The bacterial community at the dumping centre displayed a significant reduction of its entire functional diversity compared with that found at a reference site. DDX compounds separated bacterial communities of the dumping site from those of un-impacted sites. Thus, bacterial community analyses show great potential as ecological indicators in environmental monitoring.

  3. 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

  4. 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

  5. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

  6. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  7. Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos.

    PubMed Central

    Zeng, C; Pinsonneault, J; Gellon, G; McGinnis, N; McGinnis, W

    1994-01-01

    How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifically regulated by the Deformed homeotic protein. This 120 bp element (module E) is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays show that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affinity for Dfd protein generate parallel changes in the regulatory activity of module E in transgenic embryos, strong evidence that the in vitro-defined binding site is a direct target of Dfd protein in embryos. However, a monomer or multimer of the Dfd binding region alone is not sufficient to supply Dfd-dependent, segment-specific reporter gene expression. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element. The Dfd binding site and the putative cofactor binding site(s) in the region of the inverted repeat are both necessary and in combination sufficient for the function of module E. Images PMID:7910795

  8. 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.

  9. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  10. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

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

    PubMed

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

    2016-08-30

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

  12. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    PubMed Central

    Yatsenko, AS; Kucherenko, MM; Pantoja, M; Fischer, KA; Madeoy, J; Deng, W-M; Schneider, M; Baumgartner, S; Akey, J; Shcherbata, HR; Ruohola-Baker, H

    2009-01-01

    Background Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex. PMID:19250553

  13. Proteomic profiling of metalloprotease activities with cocktails of active-site probes

    PubMed Central

    Sieber, Stephan A; Niessen, Sherry; Hoover, Heather S; Cravatt, Benjamin F

    2006-01-01

    Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a ‘cocktail’ to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems. PMID:16565715

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

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

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

  15. Contributions of Unique Active Site Residues of Eukaryotic UDP-Galactopyranose Mutases to Substrate Recognition and Active Site Dynamics

    PubMed Central

    2015-01-01

    UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in kcat/KM values of 200–1000-fold for the mutase reaction. In contrast, none of the mutations significantly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together. PMID:25412209

  16. Contributions of unique active site residues of eukaryotic UDP-galactopyranose mutases to substrate recognition and active site dynamics.

    PubMed

    Da Fonseca, Isabel; Qureshi, Insaf A; Mehra-Chaudhary, Ritcha; Kizjakina, Karina; Tanner, John J; Sobrado, Pablo

    2014-12-16

    UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in k(cat)/K(M) values of 200-1000-fold for the mutase reaction. In contrast, none of the mutations significantly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together.

  17. Homology modeling and functional sites prediction of azoreductase enzyme from the cyanobacterium Nostoc sp. PCC7120.

    PubMed

    Devi, Philem Priyadarshini; Adhikari, Samrat

    2012-12-01

    Industrial dyes such as azodyes are potential environmental pollutants causing deleterious health hazards complications. These dyes are potentially degraded by azoreductase enzyme which is widely distributed in bacteria and also cyanobacteria. The azoreductase enzymes from cyanobacteria have not been explored in detail. Hence this enzyme from Nostoc sp. PCC 7120 has been addressed in detail in the present study considering to explore the physico-chemical properties, evolutionary relationships, functional sites and structural properties using various bioinformatics tools. Four conserved regions were obtained from the multiple sequence analysis. The multiple sequence alignment showed conserved regions at different stretches from 1-11, 40-57, 82-120 and 161-177 amino acid residues. These regions could be used for designing degenerate primers or probes for PCR-based amplification or hybridization-based detection of azoreductase sequences from different source organisms. Domain analysis and functional site prediction showed the presence of functional sites and domain such as flavodoxin like fold responsible for enzyme activity. 3D model was constructed and the best model was selected and validated. Superimposition of the final structure and the template showed variations in certain regions which might be involved in the accommodation of various dyes. Our results may be helpful for further investigations like docking studies as well as in vivo and in vitro conditions although these predictions still need to be studied.

  18. The active site of melanopsin: the biological clock photoreceptor.

    PubMed

    Sekharan, Sivakumar; Wei, Jennifer N; Batista, Victor S

    2012-12-05

    The nonvisual ocular photoreceptor melanopsin, found in the neurons of vertebrate inner retina, absorbs blue light and triggers the "biological clock" of mammals by activating the suprachiasmatic nuclei (a small region of the brain that regulates the circadian rhythms of neuronal and hormonal activities over 24 h cycles). The structure of melanopsin, however, has yet to be established. Here, we propose for the first time a structural model of the active site of mouse melanopsin. The homology model is based on the crystal structure of squid rhodopsin (λ(max) = 490 nm) and shows a maximal absorbance (λ(max) = 447 nm) consistent with the observed absorption of the photoreceptor. The 43 nm spectral shift is due to an increased bond-length alternation of the protonated Schiff base of 11-cis-retinal chromophore, induced by N87Q mutation and water-mediated H-bonding interactions with the Schiff base linkage. These findings, analogous to spectral changes observed in the G89Q bovine rhodopsin mutant, suggest that single site mutations can convert photopigments into visual light sensors or nonvisual sensory photoreceptors.

  19. The Hanford Site multiple function badge demonstration project

    SciTech Connect

    Nelson, R.A.

    1993-03-01

    The US Department of Energy Hanford Site is an original defense production site dating from World War II. The US Department of Energy assigned a new waste management mission for the Hanford Site. This mission requires fewer manned security posts and more areas restricted because of safety and environmental concerns. Entry into these areas requires specific safety and environmental qualifications, which could be controlled by automated access control systems. Automated entry reduces costs of managing the Site but requires health physics, safety, employee training, security databases integration, and a network system. Another approach, avoiding integration, is individuals carrying this information on their person as a picture identity badge and access control token. The health physics, safety, employee training, and security regulations that define access requirements may be best served using a smart card as the access control token. This paper discusses the need for information distribution and automated access control, potential approaches for solving the need, and smart card work at the Hanford Site.

  20. The low-affinity Ca2(+)-binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction.

    PubMed Central

    Sweeney, H L; Brito, R M; Rosevear, P R; Putkey, J A

    1990-01-01

    Both troponin C (TnC) and calmodulin share a remarkably similar tertiary motif that may be common to other Ca2(+)-binding proteins with activator activity. TnC plays a critical role in regulating muscle contraction and is particularly well-suited for structural analysis by site-directed mutation. Fast-twitch skeletal muscle TnC has two low-affinity Ca2(+)-binding sites (sites I and II), while in cardiac and slow-twitch skeletal muscle TnC site I is inactive. Recently, using protein engineering, we directly demonstrated that binding of Ca2+ to the low-affinity site(s) initiates muscle contraction. In the present study, we use mutagenesis to determine whether either of the low-affinity sites in cardiac TnC can trigger contraction in slow-twitch skeletal muscle fibers. In one Ca2(+)-binding mutant, Ca2(+)-binding to the dormant low-affinity site I was restored (CBM+I). In a second mutant, site I was activated while site II was inactivated (CBM+I-IIA). Both proteins had the predicted CA2(+)-binding characteristics, and both were able to associate with troponin I and troponin T to form a troponin complex and integrate into permeabilized slow-twitch skeletal muscle fibers. A comparison of NMR spectra shows the aromatic regions in the two proteins to be qualitatively similar without divalent cations but markedly different with Ca2+. Mutant CBM+I supported force generation in skinned slow skeletal muscle fibers but had Sr2+ and Ca2+ sensitivities similar to fast skeletal TnC. Mutant CBM+I-IIA was unable to restore Ca2(+)-dependent contraction to TnC-depleted skinned slow muscle fibers. The data directly demonstrate that low-affinity sites I and II have distinct functions and that only site II in cardiac TnC can trigger muscle contraction in slow-twitch skeletal muscle fibers. This principle of distinct, modular activities for Ca2(+)-binding sites in the same protein may apply to other members of the TnC/calmodulin family. Images PMID:2263608

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

    PubMed Central

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

    2015-01-01

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

  2. The nature of the active site in heterogeneous metal catalysis.

    PubMed

    Nørskov, Jens K; Bligaard, Thomas; Hvolbaek, Britt; Abild-Pedersen, Frank; Chorkendorff, Ib; Christensen, Claus H

    2008-10-01

    This tutorial review, of relevance for the surface science and heterogeneous catalysis communities, provides a molecular-level discussion of the nature of the active sites in metal catalysis. Fundamental concepts such as "Brønsted-Evans-Polanyi relations" and "volcano curves" are introduced, and are used to establish a strict partitioning between the so-called "electronic" and "geometrical" effects. This partitioning is subsequently employed as the basis for defining the concept "degree of structure sensitivity" which can be used when analyzing the structure sensitivity of catalytic reactions.

  3. Characterization of the active site of monkey sperm hyaluronidase.

    PubMed

    Yudin, A I; Li, M W; Robertson, K R; Cherr, G N; Overstreet, J W

    2001-05-01

    The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides representing regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the surface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molecule. Western blots of acrosome-reacted spermatozoa showed that, under reducing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also labels the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Peptide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane, fused acrosomal vesicles, acrosomal matrix and inner acrosomal membrane. Fab fragments of anti-peptide 1 IgG, but not anti-peptide 3 IgG, inhibited hyaluronidase activity of PH-20 from the sperm surface and from extracts of acrosome-reacted spermatozoa at pH 7.0. Fab fragments of both anti-peptide IgGs inhibited sperm hyaluronidase activity at pH 5.0. It is concluded that the region of PH-20 encompassed by the amino acid sequence 142-172 is essential for hyaluronidase activity at neutral pH, whereas the region of amino acid sequence 277-297 may be more important at a lower pH. It is likely that these two regions are the acid/base catalyst site and the nucleophilic site, respectively, of PH-20 hyaluronidases.

  4. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  5. Functions of Symbolizing Activity: A Discussion

    ERIC Educational Resources Information Center

    Tillema, Erik

    2010-01-01

    I propose that attending how symbolizing activity functions for teachers and students helps to characterize student-teacher communication, and allows for an investigation of how symbolizing activity contributes to learning. I begin this discussion by articulating four ideas-schemes, symbolizing activity, communication, and learning. Then I propose…

  6. 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

  7. Spectroscopic definition of the ferroxidase site in M ferritin: comparison of binuclear substrate vs cofactor active sites.

    PubMed

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

    2008-07-23

    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 nonheme 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 antiferromagnetically coupled, consistent with a mu-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.

  8. 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

  9. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

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

  10. Catalyst recognition of cis-1,2-diols enables site-selective functionalization of complex molecules

    NASA Astrophysics Data System (ADS)

    Sun, Xixi; Lee, Hyelee; Lee, Sunggi; Tan, Kian L.

    2013-09-01

    Carbohydrates and natural products serve essential roles in nature, and also provide core scaffolds for pharmaceutical agents and vaccines. However, the inherent complexity of these molecules imposes significant synthetic hurdles for their selective functionalization and derivatization. Nature has, in part, addressed these issues by employing enzymes that are able to orient and activate substrates within a chiral pocket, which increases dramatically both the rate and selectivity of organic transformations. In this article we show that similar proximity effects can be utilized in the context of synthetic catalysts to achieve general and predictable site-selective functionalization of complex molecules. Unlike enzymes, our catalysts apply a single reversible covalent bond to recognize and bind to specific functional group displays within substrates. By combining this unique binding selectivity and asymmetric catalysis, we are able to modify the less reactive axial positions within monosaccharides and natural products.

  11. Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design.

    PubMed

    Shimotohno, A; Oue, S; Yano, T; Kuramitsu, S; Kagamiyama, H

    2001-06-01

    Do non-active-site residues participate in protein function in a more direct way than just by holding the static framework of the protein molecule? If so, how important are they? As a model to answer these questions, ATB17, which is a mutant of aspartate aminotransferase created by directed evolution, is an ideal system because it shows a 10(6)-fold increase in the catalytic efficiency for valine but most of its 17 mutated residues are non-active-site residues. To analyze the roles of the mutations in the altered function, we divided the mutations into four groups, namely, three clusters and the remainder, based on their locations in the three-dimensional structure. Mutants with various combinations of the clusters were constructed and analyzed, and the data were interpreted in the context of the structure-function relationship of this enzyme. Each cluster shows characteristic effects: for example, one cluster appears to enhance the catalytic efficiency by fixing the conformation of the enzyme to that of the substrate-bound form. The effects of the clusters are largely additive and independent of each other. The present results illustrate how a protein function is dramatically modified by the accumulation of many seemingly inert mutations of non-active-site residues.

  12. Surface structural-chemical characterization of a single-site d0 heterogeneous arene hydrogenation catalyst having 100% active sites

    PubMed Central

    Williams, Linda A.; Guo, Neng; Motta, Alessandro; Delferro, Massimiliano; Fragalà, Ignazio L.; Miller, Jeffrey T.; Marks, Tobin J.

    2013-01-01

    Structural characterization of the catalytically significant sites on solid catalyst surfaces is frequently tenuous because their fraction, among all sites, typically is quite low. Here we report the combined application of solid-state 13C-cross-polarization magic angle spinning nuclear magnetic resonance (13C-CPMAS-NMR) spectroscopy, density functional theory (DFT), and Zr X-ray absorption spectroscopy (XAS) to characterize the adsorption products and surface chemistry of the precatalysts (η5-C5H5)2ZrR2 (R = H, CH3) and [η5-C5(CH3)5]Zr(CH3)3 adsorbed on Brønsted superacidic sulfated alumina (AlS). The latter complex is exceptionally active for benzene hydrogenation, with ∼100% of the Zr sites catalytically significant as determined by kinetic poisoning experiments. The 13C-CPMAS-NMR, DFT, and XAS data indicate formation of organozirconium cations having a largely electrostatic [η5-C5(CH3)5]Zr(CH3)2+···AlS− interaction with greatly elongated Zr···OAlS distances of ∼2.35(2) Å. The catalytic benzene hydrogenation cycle is stepwise understandable by DFT, and proceeds via turnover-limiting H2 delivery to surface [η5-C5(CH3)5]ZrH2(benzene)+···AlS− species, observable by solid-state NMR and XAS. PMID:23269836

  13. Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

    PubMed

    Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J; González-Feliciano, José A; Estrella, Luis A; Rodríguez-Cruz, Eva N; Correa, María E; Cajigas, Iván J; Bracho, Dina P; Vega, Irving E; Wilkinson, Miles F; González, Carlos I

    2014-02-01

    One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

  14. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  15. Synthesis and application of functional branched macromolecules: From site isolation and energy harvesting to catalysis

    NASA Astrophysics Data System (ADS)

    Hecht, Stefan

    The symbiosis of our understanding of structure property relationships in many biological macromolecules and our increased ability to prepare large synthetic macromolecules with exquisite structural precision has generated a new area of research where chemistry and materials science join with biology. For example, numerous biological systems utilize the concept of site isolation whereby an active center or catalytic site is encapsulated, frequently within a protein, to afford properties that would not be encountered in the bulk state. The ability of a dendritic shell to encapsulate functional core moieties and to create specific site-isolated nanoenvironments, thereby affecting molecular properties, not only mimics natural systems but affords novel materials with unique characteristics. Furthermore, introduction of donor chromophores at periphery of dendrimers having a central acceptor dye enables spatial and spectral energy concentration at the core. Continuing the effort towards designing bio-inspired macromolecules, this dissertation describes the use of different polymer architectures to encapsulate active sites that have either photophysical, photochemical, or catalytic functions and the evaluation of site isolation using a variety of different techniques. While the first part is mainly concerned with different synthetic approaches towards site isolation of porphyrin moieties, the second part describes the design of light-driven catalytic systems incorporating both light harvesting and energy conversion. The fundamental knowledge that can be gleaned from such investigations has implications that range from the preliminary design of artificial enzymes to the construction of molecular-scale devices. After an overview of dendritically encapsulated functions (Chapter 1) and a brief account of a novel synthetic approach to benzene core dendrimers (Chapter 2), site isolation of porphyrin moieties within dendrimers, their linear structural isomers, and branched star

  16. Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.

    PubMed

    Shu, Xiao-Gang; Su, Ji-Hu; Du, Ke-Jie; You, Yong; Gao, Shu-Qin; Wen, Ge-Bo; Tan, Xiangshi; Lin, Ying-Wu

    2016-06-01

    Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper-binding site remotely through a single mutation of Arg118 to His or Met. Isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) studies confirmed that a copper-binding site of [3-His] or [2-His-1-Met] motif was successfully created in the single mutant of R118H Mb and R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity. This study presents a new example for rational protein design with multiple active sites in a single protein scaffold, which also lays the groundwork for further investigation of the structure and function relationship of heme/non-heme proteins.

  17. Characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1.

    PubMed

    Kim, Wan-Cheol; Berquist, Brian R; Chohan, Manbir; Uy, Christopher; Wilson, David M; Lee, Chow H

    2011-09-02

    Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg(2+), while ssDNA AP site cleavage required Mg(2+) (optimally at 0.5-2.0 mM). We also found that a 2'-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3'-PO(4)(2-) group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo. Copyright

  18. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues*

    PubMed Central

    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.

    2016-01-01

    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 SeO32− 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

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

    SciTech Connect

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

    1986-05-01

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

  20. Intra-operative multi-site stimulation: Expanding methodology for cortical brain mapping of language functions

    PubMed Central

    Korn, Akiva; Kirschner, Adi; Perry, Daniella; Hendler, Talma; Ram, Zvi

    2017-01-01

    Direct cortical stimulation (DCS) is considered the gold-standard for functional cortical mapping during awake surgery for brain tumor resection. DCS is performed by stimulating one local cortical area at a time. We present a feasibility study using an intra-operative technique aimed at improving our ability to map brain functions which rely on activity in distributed cortical regions. Following standard DCS, Multi-Site Stimulation (MSS) was performed in 15 patients by applying simultaneous cortical stimulations at multiple locations. Language functioning was chosen as a case-cognitive domain due to its relatively well-known cortical organization. MSS, performed at sites that did not produce disruption when applied in a single stimulation point, revealed additional language dysfunction in 73% of the patients. Functional regions identified by this technique were presumed to be significant to language circuitry and were spared during surgery. No new neurological deficits were observed in any of the patients following surgery. Though the neuro-electrical effects of MSS need further investigation, this feasibility study may provide a first step towards sophistication of intra-operative cortical mapping. PMID:28700619

  1. Intra-operative multi-site stimulation: Expanding methodology for cortical brain mapping of language functions.

    PubMed

    Gonen, Tal; Gazit, Tomer; Korn, Akiva; Kirschner, Adi; Perry, Daniella; Hendler, Talma; Ram, Zvi

    2017-01-01

    Direct cortical stimulation (DCS) is considered the gold-standard for functional cortical mapping during awake surgery for brain tumor resection. DCS is performed by stimulating one local cortical area at a time. We present a feasibility study using an intra-operative technique aimed at improving our ability to map brain functions which rely on activity in distributed cortical regions. Following standard DCS, Multi-Site Stimulation (MSS) was performed in 15 patients by applying simultaneous cortical stimulations at multiple locations. Language functioning was chosen as a case-cognitive domain due to its relatively well-known cortical organization. MSS, performed at sites that did not produce disruption when applied in a single stimulation point, revealed additional language dysfunction in 73% of the patients. Functional regions identified by this technique were presumed to be significant to language circuitry and were spared during surgery. No new neurological deficits were observed in any of the patients following surgery. Though the neuro-electrical effects of MSS need further investigation, this feasibility study may provide a first step towards sophistication of intra-operative cortical mapping.

  2. An efficient perturbation method to predict the functionally key sites of glutamine binding protein.

    PubMed

    Lv, Dashuai; Wang, Cunxin; Li, Chunhua; Tan, Jianjun; Zhang, Xiaoyi

    2017-04-01

    Glutamine-Binding Protein (GlnBP) of Escherichia coli, an important member of the periplasmic binding protein family, is responsible for the first step in the active transport of glutamine across the cytoplasmic membrane. In this work, the functionally key regulation sites of GlnBP were identified by utilizing a perturbation method proposed by our group, in which the residues whose perturbations markedly change the binding free energy between GlnBP and glutamine are considered to be functionally key residues. The results show that besides the substrate binding sites, some other residues distant from the binding pocket, including the ones in the hinge regions between the two domains, the front- and back- door channels and the exposed region, are important for the function of glutamine binding and transport. The predicted results are well consistent with the theoretical and experimental data, which indicates that our method is an effective approach to identify the key residues important for both ligand binding and long-range allosteric signal transmission. This work can provide some insights into the function performance of GlnBP and the physical mechanism of its allosteric regulation.

  3. SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.

    PubMed

    Seamon, Kyle J; Sun, Zhiqiang; Shlyakhtenko, Luda S; Lyubchenko, Yuri L; Stivers, James T

    2015-07-27

    The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3'-5' exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. C-H Activation on Co,O Sites: Isolated Surface Sites versus Molecular Analogs.

    PubMed

    Estes, Deven P; Siddiqi, Georges; Allouche, Florian; Kovtunov, Kirill V; Safonova, Olga V; Trigub, Alexander L; Koptyug, Igor V; Copéret, Christophe

    2016-11-16

    The activation and conversion of hydrocarbons is one of the most important challenges in chemistry. Transition-metal ions (V, Cr, Fe, Co, etc.) isolated on silica surfaces are known to catalyze such processes. The mechanisms of these processes are currently unknown but are thought to involve C-H activation as the rate-determining step. Here, we synthesize well-defined Co(II) ions on a silica surface using a metal siloxide precursor followed by thermal treatment under vacuum at 500 °C. We show that these isolated Co(II) sites are catalysts for a number of hydrocarbon conversion reactions, such as the dehydrogenation of propane, the hydrogenation of propene, and the trimerization of terminal alkynes. We then investigate the mechanisms of these processes using kinetics, kinetic isotope effects, isotopic labeling experiments, parahydrogen induced polarization (PHIP) NMR, and comparison with a molecular analog. The data are consistent with all of these reactions occurring by a common mechanism, involving heterolytic C-H or H-H activation via a 1,2 addition across a Co-O bond.

  5. Endonuclease active site plasticity allows DNA cleavage with diverse alkaline Earth and transition metal ions.

    PubMed

    Vasu, Kommireddy; Saravanan, Matheshwaran; Nagaraja, Valakunja

    2011-09-16

    A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R.KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

  6. Computational approaches to find the active binding sites of biological targets against busulfan.

    PubMed

    Karthick, T; Tandon, Poonam

    2016-06-01

    Determination of electrophilic and nucleophilic sites of a molecule is the primary task to find the active sites of the lead molecule. In the present study, the active sites of busulfan have been predicted by molecular electrostatic potential surface and Fukui function analysis with the help of dispersion corrected density functional theory. Similarly, the identification of active binding sites of the proteins against lead compound plays a vital role in the field of drug discovery. Rigid and flexible molecular docking approaches are used for this purpose. For rigid docking, Hex 8.0.0 software employing fast Fourier transform (FFT) algorithm has been used. The partial flexible blind docking simulations have been performed with AutoDock 4.2 software; where a Lamarckian genetic algorithm is employed. The results showed that the most electrophilic atoms of busulfan bind with the targets. It is clear from the docking studies that busulfan has inhibition capability toward the targets 12CA and 1BZM. Graphical Abstract Docking of ligand and protein.

  7. 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.

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

    PubMed Central

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

    2016-01-01

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

  9. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. © 2012 Institute of Botany, Chinese Academy of Sciences.

  10. Functional Screening of Core Promoter Activity.

    PubMed

    Even, Dan Y; Kedmi, Adi; Ideses, Diana; Juven-Gershon, Tamar

    2017-01-01

    The core promoter is the DNA sequence that recruits the basal transcription machinery and directs accurate initiation of transcription. It is an active contributor to gene expression that can be rationally designed to manipulate the levels of expression. Core promoter function can be analyzed using different experimental approaches. Here, we describe the qualitative and quantitative analysis of engineered core promoter functions using the EGFP reporter gene that is driven by distinct core promoters. Expression plasmids are transfected into different mammalian cell lines, and the resulting fluorescence is monitored by live cell imaging , as well as by flow cytometry. In order to verify that the transcriptional activity of the examined core promoters is indeed a function of their activity, as opposed to differences in DNA uptake, real-time quantitative PCR analysis is performed. Importantly, the described methodology for functional screening of core promoter activity has enabled the analysis of engineered potent core promoters for extended time periods.

  11. Bioprospecting at former mining sites across Europe: microbial and functional diversity in soils.

    PubMed

    Sprocati, Anna Rosa; Alisi, Chiara; Tasso, Flavia; Fiore, Alessia; Marconi, Paola; Langella, Francesca; Haferburg, Götz; Nicoara, Andrei; Neagoe, Aurora; Kothe, Erika

    2014-01-01

    The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon's index (alpha diversity) and Sørensen's Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

  12. High yielding and extremely site-selective covalent functionalization of graphene.

    PubMed

    Navarro, Juan Jesús; Calleja, Fabián; Miranda, Rodolfo; Pérez, Emilio M; Vázquez de Parga, Amadeo L

    2017-09-07

    We describe a method to functionalize graphene covalently with 92% yield and 98% site-selectivity and strict spatial periodicity on the nanometer scale. This method could be extended to other functional molecules.

  13. On-Site 3D Printing of Functional Custom Mallet Splints for Mars Analogue Crewmembers.

    PubMed

    Wong, Julielynn Y

    2015-10-01

    The first off-Earth fused deposition modeling (FDM) 3D printer is investigating acrylonitrile butadiene styrene (ABS) thermoplastic manufacturing applications for long-duration space missions. This study assessed the feasibility of FDM 3D printing ABS thermoplastic customized mallet splints on site for Mars analogue crewmembers. Seven caliper measurements were taken of the right ring finger of 13 healthy Mars Desert Research Station mission crewmembers. These measurements were input into a free 3D modeling software program to create customized digital splint models. These digital files were uploaded to a desktop FDM 3D printer and custom splints were printed on site with ABS thermoplastic. Splint fit was assessed via subject feedback. Joint active range of motion was recorded when the splint was worn briefly. The time it took the software program to digitally render each splint model was less than 2 min and 30 s. The print duration for the splints ranged from 21 to 29 min. All 13 subjects reported that their 3D printed custom mallet splints fit securely and comfortably. All splints permitted full active range of motion of the proximal interphalangeal joint (0-100°) while maintaining the distal interphalangeal joint in extension. It is feasible to 3D print functional ABS thermoplastic custom mallet splints on site for Mars analogue crewmembers. This technology could be used clinically in the future when a custom mallet splint is required in a remote, resource-constrained setting.

  14. 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.

  15. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases.

  16. 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.

  17. An active site water network in the plasminogen activator pla from Yersinia pestis.

    PubMed

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

    2010-07-14

    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 A. 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.

  18. Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo

    PubMed Central

    Kaplan, Craig D.; Jin, Huiyan; Zhang, Ivan Liang; Belyanin, Andrey

    2012-01-01

    Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process. PMID:22511879

  19. Identification of functional regions on the I-Ab molecule by site-directed mutagenesis.

    PubMed Central

    Cohn, L E; Glimcher, L H; Waldmann, R A; Smith, J A; Ben-Nun, A; Seidman, J G; Choi, E

    1986-01-01

    Functional analysis of mutant class II major histocompatibility complex molecules has begun to identify regions important for antibody binding and for T-cell activation. By using in vitro mutagenesis directed at the beta 1 domain of the Ab beta gene we have constructed three structurally distinct mutant Ab beta genes. Each of these genes, as well as the wild-type Ab beta gene, was cotransfected together with the wild-type Ab alpha gene into the Ia-negative B-lymphoma cell line M12.C3. Transfection resulted in the successful synthesis and cell surface expression of three mutant class II antigens that showed serological and functional alterations as compared to the I-Ab antigens from the M12.C3 cell transfected with the wild-type gene. The variable patterns of both I-Ab-specific monoclonal antibody binding and activation of I-Ab-specific T-cell hybridomas show that the mutations result in the loss of structural epitopes required for both monoclonal antibody binding and for T-cell recognition. The data suggest that there are multiple sites on a single Ia molecule that are recognized by T helper cells and also that the tertiary conformation of the Ia molecule can be critical in the formation of such sites. PMID:2418441

  20. The structure and active site of the Varkud satellite ribozyme.

    PubMed

    Lafontaine, D A; Norman, D G; Lilley, D M

    2002-11-01

    The Varkud satellite ribozyme is the largest of the small nucleolytic ribozymes, and the only one for which there is no crystal structure. It can be divided into a trans -acting ribozyme, consisting of five helices organized by two three-way helical junctions, and a stem-loop substrate with which it interacts, primarily by tertiary interactions. We have determined the global fold of the ribozyme, and the manner by which it interacts with the substrate. The substrate interacts with a cleft formed between helices II and VI (organized by the lower helical junction), where it contacts the A730 loop, the probable active site of the ribozyme. Within this loop, there is a critical adenine base (A756) that is a candidate for direct nucleobase participation in the cleavage reaction.

  1. Characterization of the active site of ADP-ribosyl cyclase.

    PubMed

    Munshi, C; Thiel, D J; Mathews, I I; Aarhus, R; Walseth, T F; Lee, H C

    1999-10-22

    ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and

  2. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    reaction center. Synchronously, Arg366 of the β sheet at the base holds the syn oxygen of the attacking carboxylic group so that the attack by the anti oxygen is feasible. This residue also contributes to the reduction of the unfavorable electrostatic potential at the reaction center. Present simulation clearly shows the catalytic role of the residues at reaction center. A precise and stable geometry of hydrogen bonded network develops within the active site, which is essential for the development of an optimum transition state geometry. All loops move away from the platform of active site in the open or adenylate bound state and the network of hydrogen bond disappears. The serine binding site is most rigid among all three subsites. The Ser is held here in a highly organized geometry bound by Zn(2+) and Cys residues. Present simulation further suggests that the helix-turn-helix motif connecting the monomers might have important role in coordinating the functional dynamics of the two active sites. The N-terminal domain is involved in long-range electrostatic interaction and specific hydrogen bond interaction (both direct and water mediated) with tRNA. Overall conformational fluctuation is less in the N terminal compared to the catalytic domain due to the presence of a motif 2 loop, loop f, and serine ordering loop, which change conformation in the later domain during the reaction cycle. The dynamic perspective of the active site of (mk)SerRS with the mobile loop acting as the gate and dynamically silent β sheets performing as the base has similarity with the perception of the active site in various other enzymes.

  4. Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase.

    PubMed

    Ryabinina, Olga P; Minko, Irina G; Lasarev, Michael R; McCullough, Amanda K; Lloyd, R Stephen

    2011-10-10

    The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Inhibition of thrombin formation by active site mutated (S360A) activated protein C.

    PubMed

    Nicolaes, Gerry A F; Bock, Paul E; Segers, Kenneth; Wildhagen, Karin C A A; Dahlbäck, Björn; Rosing, Jan

    2010-07-23

    Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K(D) of 0.11 +/- 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.

  6. 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

  7. A method of synthesis magnetotelluric time-series combining interstation transfer functions and a reference site

    NASA Astrophysics Data System (ADS)

    Wang, Hui; Campanyà, Joan

    2016-04-01

    A new method to Synthesis magnetotelluric (MT) data is proposed whereby the MT Time-series of local site are derived using an Inverse Fourier transform of electric and magnetic fields spectrum of local site, which are computed by Combining Interstation transfer functions and a Reference horizontal magnetic time series (STICIR). The method is based on the stability of interstation transfer function, assuming that the geoelectrical structures of the subsurface independent of time. Applying the suggested method, two interstation transfer functions need to be estimated: the quasi-MT impedance tensor and the interstation geomagnetic transfer functions, which are used to compute the horizontal electric fields and the vertical magnetic field at the local site, respectively. The interstation transfer functions can be estimated by single site robust or remote reference (RR) method if another reference site existed. STICIR provides a new way to synthesis MT time-series at the local site combining interstation transfer functions and the magnetic fields at a reference site. Due to this property, STICIR provides significant improvements processing MT data when time-series at the local site are affected by local noise or are truncated. A test with good quality of MT data shows that synthetic time-series are similar to natural electric and magnetic time series. For contaminated data example, when this method is used to remove noise present at the local site, the scatter of MT sounding curves are clear reduced, and the low frequency data quality are improved.

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

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

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

  9. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  10. The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites

    PubMed Central

    West, Jason A.; Davis, Christopher P.; Sunwoo, Hongjae; Simon, Matthew D.; Sadreyev, Ruslan I.; Wang, Peggy I.; Tolstorukov, Michael Y.; Kingston, Robert E.

    2015-01-01

    Summary Mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process. PMID:25155612

  11. Predicting enzymatic function from global binding site descriptors.

    PubMed

    Volkamer, Andrea; Kuhn, Daniel; Rippmann, Friedrich; Rarey, Matthias

    2013-03-01

    Due to the rising number of solved protein structures, computer-based techniques for automatic protein functional annotation and classification into families are of high scientific interest. DoGSiteScorer automatically calculates global descriptors for self-predicted pockets based on the 3D structure of a protein. Protein function predictors on three levels with increasing granularity are built by use of a support vector machine (SVM), based on descriptors of 26632 pockets from enzymes with known structure and enzyme classification. The SVM models represent a generalization of the available descriptor space for each enzyme class, subclass, and substrate-specific sub-subclass. Cross-validation studies show accuracies of 68.2% for predicting the correct main class and accuracies between 62.8% and 80.9% for the six subclasses. Substrate-specific recall rates for a kinase subset are 53.8%. Furthermore, application studies show the ability of the method for predicting the function of unknown proteins and gaining valuable information for the function prediction field. Copyright © 2012 Wiley Periodicals, Inc.

  12. Biological growth functions describe published site index curves for Lake States timber species.

    Treesearch

    Allen L. Lundgren; William A. Dolid

    1970-01-01

    Two biological growth functions, an exponential-monomolecular function and a simple monomolecular function, have been fit to published site index curves for 11 Lake States tree species: red, jack, and white pine, balsam fir, white and black spruce, tamarack, white-cedar, aspen, red oak, and paper birch. Both functions closely fit all published curves except those for...

  13. Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants.

    PubMed Central

    Roberts, G.; Berberian, G.; Beauge, L.

    1995-01-01

    We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases. To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis thaliana. The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast. ATP stimulation was always with low affinity (K0.5 between 500 and 1800 [mu]M). In addition, the curves were not Michaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kinetics competence of the E(ATP) and E(ATP)2 complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule. PMID:12228512

  14. Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution

    PubMed Central

    Campanini, Barbara; 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. PMID:23984351

  15. A unique geometry of the active site of angiotensin-converting enzyme consistent with structure-activity studies

    NASA Astrophysics Data System (ADS)

    Mayer, Dorica; Naylor, Christopher B.; Motoc, Ioan; Marshall, Garland R.

    1987-04-01

    Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 Å) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

  16. Bent Carbon Surface Moieties as Active Sites on Carbon Catalysts for Phosgene Synthesis.

    PubMed

    Gupta, Navneet K; Pashigreva, Anastasia; Pidko, Evgeny A; Hensen, Emiel J M; Mleczko, Leslaw; Roggan, Stefan; Ember, Erika E; Lercher, Johannes A

    2016-01-26

    Active sites in carbon-catalyzed phosgene synthesis from gaseous CO and Cl2 have been identified using C60 fullerene as a model catalyst. The carbon atoms distorted from sp(2) coordination in non-planar carbon units are concluded to generate active Cl2 . Experiments and density functional theory calculations indicate the formation of a surface-bound [C60 ⋅⋅⋅Cl2 ] chlorine species with radical character as key intermediate during phosgene formation. It reacts rapidly with physisorbed CO in a two-step Eley-Rideal-type mechanism. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Structure/Function/Dynamics of Photosystem II Plastoquinone Binding Sites

    PubMed Central

    Lambreva, Maya D.; Russo, Daniela; Polticelli, Fabio; Scognamiglio, Viviana; Antonacci, Amina; Zobnina, Veranika; Campi, Gaetano; Rea, Giuseppina

    2014-01-01

    Photosystem II (PSII) continuously attracts the attention of researchers aiming to unravel the riddle of its functioning and efficiency fundamental for all life on Earth. Besides, an increasing number of biotechnological applications have been envisaged exploiting and mimicking the unique properties of this macromolecular pigment-protein complex. The PSII organization and working principles have inspired the design of electrochemical water splitting schemes and charge separating triads in energy storage systems as well as biochips and sensors for environmental, agricultural and industrial screening of toxic compounds. An intriguing opportunity is the development of sensor devices, exploiting native or manipulated PSII complexes or ad hoc synthesized polypeptides mimicking the PSII reaction centre proteins as bio-sensing elements. This review offers a concise overview of the recent improvements in the understanding of structure and function of PSII donor side, with focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. Furthermore, studies focused on photosynthetic proteins structure/function/dynamics and computational analyses aimed at rational design of high-quality bio-recognition elements in biosensor devices are discussed. PMID:24678671

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

    PubMed

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

    2016-08-01

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

  19. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  20. Mercury Binding Sites in Thiol-Functionalized Mesostructured Silica

    SciTech Connect

    Billinge, Simon J.L.; McKimmey, Emily J.; Shatnawi, Mouath; Kim, HyunJeong; Petkov, Valeri; Wermeille, Didier; Pinnavaia, Thomas J.

    2010-07-13

    Thiol-functionalized mesostructured silica with anhydrous compositions of (SiO{sub 2}){sub 1-x}(LSiO{sub 1.5}){sub x}, where L is a mercaptopropyl group and x is the fraction of functionalized framework silicon centers, are effective trapping agents for the removal of mercuric(II) ions from water. In the present work, we investigate the mercury-binding mechanism for representative thiol-functionalized mesostructures by atomic pair distribution function (PDF) analysis of synchrotron X-ray powder diffraction data and by Raman spectroscopy. The mesostructures with wormhole framework structures and compositions corresponding to x = 0.30 and 0.50 were prepared by direct assembly methods in the presence of a structure-directing amine porogen. PDF analyses of five mercury-loaded compositions with Hg/S ratios of 0.50-1.30 provided evidence for the bridging of thiolate sulfur atoms to two metal ion centers and the formation of chain structures on the pore surfaces. We find no evidence for Hg-O bonds and can rule out oxygen coordination of the mercury at greater than the 10% level. The relative intensities of the PDF peaks corresponding to Hg-S and Hg-Hg atomic pairs indicate that the mercury centers cluster on the functionalized surfaces by virtue of thiolate bridging, regardless of the overall mercury loading. However, the Raman results indicate that the complexation of mercury centers by thiolate depends on the mercury loading. At low mercury loadings (Hg/S {le} 0.5), the dominant species is an electrically neutral complex in which mercury most likely is tetrahedrally coordinated to bridging thiolate ligands, as in Hg(SBu{sup t}){sub 2}. At higher loadings (Hg/S 1.0-1.3), mercury complex cations predominate, as evidenced by the presence of charge-balancing anions (nitrate) on the surface. This cationic form of bound mercury is assigned a linear coordination to two bridging thiolate ligands.

  1. Functional activity monitoring from wearable sensor data.

    PubMed

    Nawab, S Hamid; Roy, Serge H; De Luca, Carlo J

    2004-01-01

    A novel approach is presented for the interpretation and use of EMG and accelerometer data to monitor, identify, and categorize functional motor activities in individuals whose movements are unscripted, unrestrained, and take place in the "real world". Our proposed solution provides a novel and practical way of conceptualizing physical activities that facilitates the deployment of modern signal processing and interpretation techniques to carry out activity monitoring. A hierarchical approach is adopted that is based upon: 1) blackboard and rule-based technology from artificial intelligence to support a process in which coarse-grained activity partitioning forms the context for finer-grained activity partitioning; 2) neural network technology to support initial activity classification; and 3) integrated processing and understanding of signals (IPUS) technology for revising the initial classifications to account for the high degrees of anticipated signal variability and overlap during freeform activity.

  2. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

    PubMed Central

    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.

    2016-01-01

    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. PMID:27581526

  3. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  4. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  5. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  6. Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine.

    PubMed

    Liu, Monica Yun; Torabifard, Hedieh; Crawford, Daniel J; DeNizio, Jamie E; Cao, Xing-Jun; Garcia, Benjamin A; Cisneros, G Andrés; Kohli, Rahul M

    2017-02-01

    Ten-eleven translocation (TET) enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) to yield 5-hydroxymethylcytosine (hmC) and the rarer bases 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Stepwise oxidation obscures how each individual base forms and functions in epigenetic regulation, and prompts the question of whether TET enzymes primarily serve to generate hmC or are adapted to produce fC and caC as well. By mutating a single, conserved active site residue in human TET2, Thr1372, we uncovered enzyme variants that permit oxidation to hmC but largely eliminate fC and caC. Biochemical analyses, combined with molecular dynamics simulations, elucidated an active site scaffold that is required for wild-type (WT) stepwise oxidation and that, when perturbed, explains the mutants' hmC-stalling phenotype. Our results suggest that the TET2 active site is shaped to enable higher-order oxidation and provide the first TET variants that could be used to probe the biological functions of hmC separately from fC and caC.

  7. Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

    PubMed Central

    Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

    2012-01-01

    Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747

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

    SciTech Connect

    Gopal, B.; Madan, Lalima L.; Betz, Stephen F.; Kossiakoff, Anthony A.

    2010-11-10

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

  9. SITEX 2.0: Projections of protein functional sites on eukaryotic genes. Extension with orthologous genes.

    PubMed

    Medvedeva, Irina V; Demenkov, Pavel S; Ivanisenko, Vladimir A

    2017-04-01

    Functional sites define the diversity of protein functions and are the central object of research of the structural and functional organization of proteins. The mechanisms underlying protein functional sites emergence and their variability during evolution are distinguished by duplication, shuffling, insertion and deletion of the exons in genes. The study of the correlation between a site structure and exon structure serves as the basis for the in-depth understanding of sites organization. In this regard, the development of programming resources that allow the realization of the mutual projection of exon structure of genes and primary and tertiary structures of encoded proteins is still the actual problem. Previously, we developed the SitEx system that provides information about protein and gene sequences with mapped exon borders and protein functional sites amino acid positions. The database included information on proteins with known 3D structure. However, data with respect to orthologs was not available. Therefore, we added the projection of sites positions to the exon structures of orthologs in SitEx 2.0. We implemented a search through database using site conservation variability and site discontinuity through exon structure. Inclusion of the information on orthologs allowed to expand the possibilities of SitEx usage for solving problems regarding the analysis of the structural and functional organization of proteins. Database URL: http://www-bionet.sscc.ru/sitex/ .

  10. Mapping solvation dynamics at the function site of flavodoxin in three redox states.

    PubMed

    Chang, Chih-Wei; He, Ting-Fang; Guo, Lijun; Stevens, Jeffrey A; Li, Tanping; Wang, Lijuan; Zhong, Dongping

    2010-09-15

    Flavoproteins are unique redox coenzymes, and the dynamic solvation at their function sites is critical to the understanding of their electron-transfer properties. Here, we report our complete characterization of the function-site solvation of holoflavodoxin in three redox states and of the binding-site solvation of apoflavodoxin. Using intrinsic flavin cofactor and tryptophan residue as the local optical probes with two site-specific mutations, we observed distinct ultrafast solvation dynamics at the function site in the three states and at the related recognition site of the cofactor, ranging from a few to hundreds of picoseconds. The initial ultrafast motion in 1-2.6 ps reflects the local water-network relaxation around the shallow, solvent-exposed function site. The second relaxation in 20-40 ps results from the coupled local water-protein fluctuation. The third dynamics in hundreds of picoseconds is from the intrinsic fluctuation of the loose loops flanking the cofactor at the function site. These solvation dynamics with different amplitudes well correlate with the redox states from the oxidized form, to the more rigid semiquinone and to the much looser hydroquinone. This observation of the redox control of local protein conformation plasticity and water network flexibility is significant, and such an intimate relationship is essential to the biological function of interprotein electron transfer.

  11. Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.

    PubMed

    Steegborn, Clemens; Litvin, Tatiana N; Levin, Lonny R; Buck, Jochen; Wu, Hao

    2005-01-01

    In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3',5'-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.

  12. A split active site couples cap recognition by Dcp2 to activation

    PubMed Central

    Floor, Stephen N.; Jones, Brittnee N.; Hernandez, Gail A.; Gross, John D.

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a factor of ten and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping, where coactivators trigger decapping by stabilizing a labile composite active site. PMID:20711189

  13. L2' loop is critical for caspase-7 active site formation.

    PubMed

    Witkowski, Witold A; Hardy, Jeanne A

    2009-07-01

    The active sites of caspases are composed of four mobile loops. A loop (L2) from one half of the dimer interacts with a loop (L2') from the other half of the dimer to bind substrate. In an inactive form, the two L2' loops form a cross-dimer hydrogen-bond network over the dimer interface. Although the L2' loop has been implicated as playing a central role in the formation of the active-site loop bundle, its precise role in catalysis has not been shown. A detailed understanding of the active and inactive conformations is essential to control the caspase function. We have interrogated the contributions of the residues in the L2' loop to catalytic function and enzyme stability. In wild-type and all mutants, active-site binding results in substantial stabilization of the complex. One mutation, P214A, is significantly destabilized in the ligand-free conformation, but is as stable as wild type when bound to substrate, indicating that caspase-7 rests in different conformations in the absence and presence of substrate. Residues K212 and I213 in the L2' loop are shown to be essential for substrate-binding and thus proper catalytic function of the caspase. In the crystal structure of I213A, the void created by side-chain deletion is compensated for by rearrangement of tyrosine 211 to fill the void, suggesting that the requirements of substrate-binding are sufficiently strong to induce the active conformation. Thus, although the L2' loop makes no direct contacts with substrate, it is essential for buttressing the substrate-binding groove and is central to native catalytic efficiency.

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

    PubMed

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

    2013-08-01

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

  15. Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3.

    PubMed

    Shen, Hujun; Deng, Mingsen; Zhang, Yachao

    2017-09-12

    Recent crystal structures of RNA-dependent RNA polymerase (3D(pol)) from Coxsackievirus B3 (CVB3) revealed that a tyrosine mutation at Phe364 (F364Y) resulted in structures with open active site whereas a hydrophobic mutation at Phe364 (F364A) led to conformations with closed active site. Besides, the crystal structures showed that the F364W mutation had no preference between the open and closed active sites, similar to wild-type. In this paper, we present a molecular dynamics (MD) study on CVB3 3D(pol) in order to address some important questions raised by experiments. First, MD simulations of F364Y and F364A were carried out to explore how these mutations at Phe364 influence active site dynamics and conformations. Second, MD simulations of wild-type and mutants were performed to discover the connection between active site dynamics and polymerase function. MD simulations reveal that the effect of mutations on active site dynamics is associated with the interaction between the structural motifs A and D in CVB3 3D(pol). Interestingly, we discover that the active site state is influenced by the formation of a hydrogen bond between backbone atoms of Ala231 (in motif A) and Ala358 (in motif D), which has never been revealed before. Copyright © 2017. Published by Elsevier Inc.

  16. Ligand binding to the AMP-activated protein kinase active site mediates protection of the activation loop from dephosphorylation.

    PubMed

    Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; Schmidt, Martin C

    2013-01-04

    The AMP-activated protein kinase (AMPK) is a conserved signaling molecule in a pathway that maintains adenosine triphosphate homeostasis. Recent studies have suggested that low energy adenylate ligands bound to one or more sites in the γ subunit of AMPK promote the formation of an active, phosphatase-resistant conformation. We propose an alternative model in which the kinase domain association with the heterotrimer core results in activation of the kinase catalytic activity, whereas low energy adenylate ligands bound in the kinase active site promote phosphatase resistance. Purified Snf1 α subunit with a conservative, single amino acid substitution in the kinase domain is protected from dephosphorylation by adenosine diphosphate in the complete absence of the β and γ subunits. Staurosporine, a compound known to bind to the active site of many protein kinases, mediates strong protection from dephosphorylation to yeast and mammalian AMPK enzymes. The analog-sensitive Snf1-I132G protein but not wild type Snf1 exhibits protection from dephosphorylation when bound by the adenosine analog 2NM-PP1 in vitro and in vivo. These data demonstrate that ligand binding to the Snf1 active site can mediate phosphatase resistance. Finally, Snf1 kinase with an amino acid substitution at the interface of the kinase domain and the heterotrimer core exhibits normal regulation of phosphorylation in vivo but greatly reduced Snf1 kinase activity, supporting a model in which kinase domain association with the heterotrimer core is needed for kinase activation.

  17. Toward a Functional Definition of Methane Super-Emitters: Application to Natural Gas Production Sites.

    PubMed

    Zavala-Araiza, Daniel; Lyon, David; Alvarez, Ramón A; Palacios, Virginia; Harriss, Robert; Lan, Xin; Talbot, Robert; Hamburg, Steven P

    2015-07-07

    Emissions from natural gas production sites are characterized by skewed distributions, where a small percentage of sites-commonly labeled super-emitters-account for a majority of emissions. A better characterization of super-emitters is needed to operationalize ways to identify them and reduce emissions. We designed a conceptual framework that functionally defines superemitting sites as those with the highest proportional loss rates (methane emitted relative to methane produced). Using this concept, we estimated total methane emissions from natural gas production sites in the Barnett Shale; functionally superemitting sites accounted for roughly three-fourths of total emissions. We discuss the potential to reduce emissions from these sites, under the assumption that sites with high proportional loss rates have excess emissions resulting from abnormal or otherwise avoidable operating conditions, such as malfunctioning equipment. Because the population of functionally superemitting sites is not expected to be static over time, continuous monitoring will likely be necessary to identify them and improve their operation. This work suggests that achieving and maintaining uniformly low emissions across the entire population of production sites will require mitigation steps at a large fraction of sites.

  18. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    SciTech Connect

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E.

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  19. Targeting the Binding Function 3 (BF3) Site of the Human Androgen Receptor Through Virtual Screening

    PubMed Central

    Lack, Nathan A.; Axerio-Cilies, Peter; Tavassoli, Peyman; Han, Frank Q.; Chan, Ka Hong; Feau, Clementine; LeBlanc, Eric; Guns, Emma Tomlinson; Guy, R. Kiplin; Rennie, Paul S.; Cherkasov, Artem

    2013-01-01

    The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called Binding Function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in-silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and x-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its anti-androgen resistant forms. PMID:22047606

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

    PubMed

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

    2011-07-01

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

  1. Controlling activation site density by low-energy far-field stimulation in cardiac tissue

    NASA Astrophysics Data System (ADS)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites (“virtual electrodes”) in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  2. Controlling activation site density by low-energy far-field stimulation in cardiac tissue.

    PubMed

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites ("virtual electrodes") in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  3. The enzymes of the transsulfuration pathways: active-site characterizations.

    PubMed

    Aitken, Susan M; Lodha, Pratik H; Morneau, Dominique J K

    2011-11-01

    The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5'-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine β-lyase) and reverse transsulfuration (cystathionine β-synthase and cystathionine γ-lyase) pathways interconvert l-cysteine and l-homocysteine, the immediate precursor of l-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology. Copyright © 2011. Published by Elsevier B.V.

  4. 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.

  5. 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.

  6. Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase.

    PubMed

    Mercaldi, Gustavo F; Pereira, Humberto M; Cordeiro, Artur T; Michels, Paul A M; Thiemann, Otavio H

    2012-06-01

    Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.

  7. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    SciTech Connect

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  8. CO-dynamics in the active site of cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Soloviov, Maksym; Meuwly, Markus

    2014-04-01

    The transfer of CO from heme a3 to the CuB site in Cytochrome c oxidase (CcO) after photolysis is studied using molecular dynamics simulations using an explicitly reactive, parametrized potential energy surface based on density functional theory calculations. After photodissociation from the heme-Fe, the CO ligand rebinds to the CuB site on the sub-picosecond time scale. Depending on the simulation protocol the characteristic time ranges from 260 fs to 380 fs which compares with an estimated 450 fs from experiment based on the analysis of the spectral changes as a function of time delay after the photodissociating pulse. Following photoexcitation ≈90% of the ligands are found to rebind to either the CuB (major component, 85%) or the heme-Fe (minor component, 2%) whereas about 10% remain in an unbound state. The infrared spectra of unbound CO in the active site is broad and featureless and no appreciable shift relative to gas-phase CO is found, which is in contrast to the situation in myoglobin. These observations explain why experimentally, unbound CO in the binuclear site of CcO has not been found as yet.

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

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

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

  10. 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.

  11. Opiates and cerebral functional activity in rats

    SciTech Connect

    Trusk, T.C.

    1986-01-01

    Cerebral activity was measured using the free-fatty acid (1-/sup 14/C) octanoate as a fast functional tracer in conscious, unrestrained rats 5 minutes after intravenous injection of heroin, cocaine or saline vehicle. Regional changes of octanoate labeling density in the autoradiograms relative to saline-injected animals were used to determine the functional activity effects of each drug. Heroin and cocaine each produced a distinctive pattern of activity increases and suppression throughout the rat brain. Similar regional changes induced by both drugs were found in limbic brain regions implicated in drug reinforcement. Labeled octanoate autoradiography was used to measure the cerebral functional response to a tone that had previously been paired to heroin injections. Rats were trained in groups of three consisting of one heroin self-administration animal, and two animals receiving yoked infusion of heroin or saline. A tone was paired with each infusion during training. Behavioral experiments in similarly trained rats demonstrated that these training conditions impart secondary reinforcing properties to the tone in animals previously self-administering heroin, while the tone remains behaviorally neutral in yoked-infusion rats. Cerebral functional activity was measured during presentation of the tone without drug infusion. Octanoate labeling density changed in fifteen brain areas in response to the tone previously paired to heroin without response contingency. Labeling density was significantly modified in sixteen regions as a result of previously pairing the tone to response-contingent heroin infusions.

  12. Cerebral blood flow response to functional activation

    PubMed Central

    Paulson, Olaf B; Hasselbalch, Steen G; Rostrup, Egill; Knudsen, Gitte Moos; Pelligrino, Dale

    2010-01-01

    Cerebral blood flow (CBF) and cerebral metabolic rate are normally coupled, that is an increase in metabolic demand will lead to an increase in flow. However, during functional activation, CBF and glucose metabolism remain coupled as they increase in proportion, whereas oxygen metabolism only increases to a minor degree—the so-called uncoupling of CBF and oxidative metabolism. Several studies have dealt with these issues, and theories have been forwarded regarding the underlying mechanisms. Some reports have speculated about the existence of a potentially deficient oxygen supply to the tissue most distant from the capillaries, whereas other studies point to a shift toward a higher degree of non-oxidative glucose consumption during activation. In this review, we argue that the key mechanism responsible for the regional CBF (rCBF) increase during functional activation is a tight coupling between rCBF and glucose metabolism. We assert that uncoupling of rCBF and oxidative metabolism is a consequence of a less pronounced increase in oxygen consumption. On the basis of earlier studies, we take into consideration the functional recruitment of capillaries and attempt to accommodate the cerebral tissue's increased demand for glucose supply during neural activation with recent evidence supporting a key function for astrocytes in rCBF regulation. PMID:19738630

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

    PubMed

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

    2016-09-01

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

  14. 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

    2014-06-02

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Functional activities of the amygdala: an overview.

    PubMed Central

    Rasia-Filho, A A; Londero, R G; Achaval, M

    2000-01-01

    Research to date into the amygdala shows that it has an integrative role in behavioural, vegetative and endocrine activities of animals in their relation with their environment. Animal studies show that amygdala has a role in emotional response, integrating input signals and initiating activities related to them. Different nuclei seem to have different effects. A complete picture of the functional roles of the amygdala is unavailable, and it has been suggested that the amygdala is functionally and anatomically heterogeneous. Amygdaloid subnuclei appear to have a role in the modulation of fear, in memory and attention, and in some sexual and sex-related behaviour of rats. In humans, functional magnetic resonance imaging shows that the amygdala responds preferentially to emotionally charged stimuli. Bilateral amygdala damage in humans can compromise the recognition of fear in facial expressions, an important ability in social judgement. Future study of the amygdala promises to shed light on emotional disorders in humans. PMID:10721680

  16. Pseudo-active sites of protease domains: HGF/Met and Sonic hedgehog signaling in cancer.

    PubMed

    Maun, Henry R; Kirchhofer, Daniel; Lazarus, Robert A

    2010-08-01

    Proteases represent a large class of enzymes with crucial biological functions. Although targeting various relevant proteases for therapeutic intervention has been widely investigated, structurally related proteins lacking proteolytic activity (pseudo-proteases) have received relatively little attention. Two distinct clinically relevant cancer pathways that contain signaling proteins with pseudo-protease domains include the Met and Hedgehog (Hh) pathways. The receptor tyrosine kinase Met pathway is driven by hepatocyte growth factor (HGF), a plasminogen-related ligand that binds Met and activates intracellular pathways resulting in cell proliferation, angiogenesis, motility and survival. HGF is a disulfide-linked alpha/beta-heterodimer having a trypsin serine protease-like beta-chain. The Hh pathway is driven by Sonic hedgehog (Shh), which has a Zn(2+) metalloprotease fold and binds Patched1 (Ptc1), which de-represses Smoothened and ultimately activates Gli-dependent transcription. Although HGF and Shh differ in structure and function, the pseudo-catalytic sites of both HGF and Shh are crucial for signal transduction. For HGF, this region binds the Met beta-propeller domain, which leads to Met dimerization and signaling. For Hh, this region binds to the antagonist receptor Hedgehog-interacting protein (Hhip) and most probably to Ptc1 as well. Thus, for both HGF and Hh pathways, targeting ligand pseudo-active sites represents a new strategy for regulation.

  17. Mechanism and Site Selectivity in Visible-Light Photocatalyzed C-H Functionalization: Insights from DFT Calculations.

    PubMed

    Demissie, Taye B; Hansen, Jørn H

    2016-08-19

    Visible-light photocatalyzed (VLPC) late-stage C-H functionalization is a powerful addition to the chemical synthesis toolkit. VLPC has a demonstrated potential for discovery of elusive and valuable transformations, particularly in functionalization of bioactive heterocycles. In order to fully harvest the potential of VLPC in the context of complex molecule synthesis, a thorough understanding of the elementary processes involved is crucial. This would enable more rational design of suitable reagents and catalysts, as well as prediction of activated C-H sites for functionalization. Such knowledge is essential when VLPC is to be employed in retrosynthetic analysis of complex molecules. Herein, we present a density functional theory (DFT) study of mechanistic details in the C-H functionalization of bioactive heterocycles exemplified by the methylation of the antifungal agent voriconazole. Moreover, we show that readily computed atomic charges can predict major site-selectivity in good agreement with experimental studies and thus be informative tools for the identification of active C-H functionalization sites in synthetic planning.

  18. Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality*

    PubMed Central

    Žáková, Lenka; Kletvíková, Emília; Veverka, Václav; Lepšík, Martin; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2013-01-01

    Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant PheB24, one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to d-amino acids but not to l-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [d-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the l-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [d-HisB24]-insulin is more flexible, and the reverse chirality of the B24Cα atom swayed the d-HisB24 side chain into the solvent. Furthermore, the pocket vacated by PheB24 is filled by PheB25, which mimics the PheB24 side and main chains. The B25→B24 downshift results in a subsequent downshift of TyrB26 into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic l-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation. PMID:23447530

  19. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

    PubMed Central

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  20. Site-selective and stereoselective functionalization of unactivated C-H bonds

    NASA Astrophysics Data System (ADS)

    Liao, Kuangbiao; Negretti, Solymar; Musaev, Djamaladdin G.; Bacsa, John; Davies, Huw M. L.

    2016-05-01

    The laboratory synthesis of complex organic molecules relies heavily on the introduction and manipulation of functional groups, such as carbon-oxygen or carbon-halogen bonds; carbon-hydrogen bonds are far less reactive and harder to functionalize selectively. The idea of C-H functionalization, in which C-H bonds are modified at will instead of the functional groups, represents a paradigm shift in the standard logic of organic synthesis. For this approach to be generally useful, effective strategies for site-selective C-H functionalization need to be developed. The most practical solutions to the site-selectivity problem rely on either intramolecular reactions or the use of directing groups within the substrate. A challenging, but potentially more flexible approach, would be to use catalyst control to determine which site in a particular substrate would be functionalized. Here we describe the use of dirhodium catalysts to achieve highly site-selective, diastereoselective and enantioselective C-H functionalization of n-alkanes and terminally substituted n-alkyl compounds. The reactions proceed in high yield, and functional groups such as halides, silanes and esters are compatible with this chemistry. These studies demonstrate that high site selectivity is possible in C-H functionalization reactions without the need for a directing or anchoring group present in the molecule.

  1. 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.

  2. Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism.

    PubMed

    Mones, Letif; Simon, István; Fuxreiter, Monika

    2007-01-01

    The number of metal ions required for phosphoryl transfer in restriction endonucleases is still an unresolved question in molecular biology. The two Ca(2+) and Mn(2+) ions observed in the pre- and post-reactive complexes of BamHI conform to the classical two-metal ion choreography. We probed the Mg(2+) cofactor positions at the active site of BamHI by molecular dynamics simulations with one and two metal ions present and identified several catalytically relevant sites. These can mark the pathway of a single ion during catalysis, suggesting its critical role, while a regulatory function is proposed for a possible second ion.

  3. PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei.

    PubMed

    Urményi, T P; Van der Ploeg, L H

    1995-03-25

    In trypanosomes the rRNA, PARP and VSG gene promoters mediate alpha-amanitin-resistant transcription of protein coding genes, presumably by RNA polymerase (pol) I. We compared the activity of PARP and VSG promoters integrated at one of the alleles of the largest subunit of pol II genes in insect form trypanosomes. Even though both promoters are roughly equally active in transient transformation assays in insect form trypanosomes, only the PARP promoter functioned effectively when integrated at the pol II largest subunit or other loci. Promoter activity in transient transformation assays is therefore not necessarily predictive of transcriptional activity once integrated into the trypanosome genome. The integrated fully active PARP promoter could upregulate in cis an otherwise poorly active integrated VSG promoter. The PARP promoter nucleotide sequence elements responsible for VSG promoter activation coincided with most of the important PARP promoter elements mapped previously by linker scanning mutagenesis, indicating that it is not a single unique promoter element that was responsible for VSG promoter activation. The data suggest that PARP promoter-mediated activation of the VSG promoter does not result from complementation of the VSG promoter with a single insect form-specific transcription factor whose binding site is missing from the VSG promoter and present in the PARP promoter. We favor a model in which chromatin structure at the locus is altered by the PARP promoter, allowing VSG promoter activation in insect form trypanosomes. We discuss the significance of these observations for the control of VSG promoters in insect form trypanosomes.

  4. A discrimlnant function approach to ecological site classification in northern New England

    Treesearch

    James M. Fincher; Marie-Louise Smith

    1994-01-01

    Describes one approach to ecologically based classification of upland forest community types of the White and Green Mountain physiographic regions. The classification approach is based on an intensive statistical analysis of the relationship between the communities and soil-site factors. Discriminant functions useful in distinguishing between types based on soil-site...

  5. Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors

    PubMed Central

    Estrada-Mondragon, Argel; Lynch, Joseph W.

    2015-01-01

    GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36′F sidechains to the “+” side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36′ methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites. PMID:26441518

  6. Rna-seq analysis of the functional compartments within the rat placentation site

    USDA-ARS?s Scientific Manuscript database

    The rat placentation site is distinctly organized into interacting zones, the so-called labyrinth, junctional, and metrial gland compartments. These zones house unique cell populations equipped to undertake myriad prescribed functions including transport, hormonal responses, and immune interactions....

  7. Predicting functional divergence in protein evolution by site-specific rate shifts

    NASA Technical Reports Server (NTRS)

    Gaucher, Eric A.; Gu, Xun; Miyamoto, Michael M.; Benner, Steven A.

    2002-01-01

    Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the "non-homogeneous gamma model", which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

  8. Predicting functional divergence in protein evolution by site-specific rate shifts

    NASA Technical Reports Server (NTRS)

    Gaucher, Eric A.; Gu, Xun; Miyamoto, Michael M.; Benner, Steven A.

    2002-01-01

    Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the "non-homogeneous gamma model", which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

  9. 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.

  10. XIAP reverses various functional activities of FRNK in endothelial cells

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  11. 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 at...

  12. 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 at...

  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 at...

  14. 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 at...

  15. 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 at...

  16. Lidar research activities and observations at NARL site, Gadanki, India

    NASA Astrophysics Data System (ADS)

    Yellapragada, Bhavani Kumar

    2016-05-01

    The National Atmospheric Research Laboratory (NARL), a unit of Department of Space (DOS), located at Gadanki village (13.5°N, 79.2°E, 370 m AMSL) in India, is involved in the development of lidar remote sensing technologies for atmospheric research. Several advanced lidar technologies employing micropulse, polarization, Raman and scanning have been developed at this site and demonstrated for atmospheric studies during the period between 2008 and 2015. The technology of micropulse lidar, operates at 532 nm wavelength, was successfully transferred to an industry and the commercial version has been identified for Indian Lidar network (I-LINK) programme. Under this lidar network activity, several lidar units were installed at different locations in India to study tropospheric aerosols and clouds. The polarization sensitive lidar technology was realized using a set of mini photomultiplier tube (PMT) units and has the capability to operate during day and night without a pause. The lidar technology uses a compact flashlamp pumped Qswitched laser and employs biaxial configuration between the transmitter and receiver units. The lidar technology has been utilized for understanding the polarization characteristics of boundary layer aerosols during the mixed layer development. The demonstrated Raman lidar technology, uses the third harmonic wavelength of Nd:YAG laser, provides the altitude profiles of aerosol backscattering, extinction and water vapor covering the boundary layer range and allows operation during nocturnal periods. The Raman lidar derived height profiles of aerosol backscattering and extinction coefficient, lidar ratio, and watervapor mixing ratio inform the tropical boundary layer aerosol characteristics. The scanning lidar technology uses a near infrared laser wavelength for probing the lower atmosphere and has been utilized for high resolution cloud profiling during convective periods. The lidar technology is also used for rain rate measurement during

  17. 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.

  18. Stochastic analysis of myoelectric temporal signatures for multifunctional single-site activation of prostheses and orthoses.

    PubMed

    Graupe, D; Salahi, J; Zhang, D S

    1985-01-01

    This paper is concerned with a stochastic time-series analysis of the temporal signatures of myoelectric (ME) signals including the determination of model order and sampling rate. The paper considers the use of time-series parameters for the activation of artificial limbs for high-level amputees, of stimulation electrodes or of powered braces for paralysed persons, in several degrees of freedom, from a single or two surface-electrode pairs at locations where considerable ME cross-talk exists. The multifunctional capability from a single site is based on the differences between the time-series (TS) parameters for different muscle activation patterns at the same ME site, these differences being thus used for limb function discrimination via easily trainable muscle activation patterns at the vicinity of the electrode site. Specifically, the analysis is in terms of identifying the AR parameters of a time-domain autoregressive (AR) signature model both for the complete ME spectrum and for parts thereof, and in terms of the autocorrelation of the signal and of the models residual. Determination of sampling rate and of model orders is discussed in detail. It is shown that, using online real-time analysis, differences in the AR time-series parameters can be observed for different trainable patterns of muscle activation, at the same electrode location, even at the same ME power levels, as long as considerable cross-talk exists at the electrode site. These parameter differences can be accentuated if one considers the AR parameters for lower-frequency spectral windows. A case is made in this paper for employing TS analysis to squeeze out information in a distinct but low-level ripple of the low frequency spectrum of the signal. This information tends to be ignored in frequency domain, but is all that the AR parameters car