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Sample records for acceptor substrate binding

  1. Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1.

    PubMed

    Wang, Juan; Liu, Xiang; Liang, Yu-He; Li, Lan-Fen; Su, Xiao-Dong

    2008-09-01

    Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. PMID:18675810

  2. Probing the donor and acceptor substrate specificity of the γ-glutamyl transpeptidase.

    PubMed

    Hu, Xin; Legler, Patricia M; Khavrutskii, Ilja; Scorpio, Angelo; Compton, Jaimee R; Robertson, Kelly L; Friedlander, Arthur M; Wallqvist, Anders

    2012-02-14

    γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors. PMID:22257032

  3. Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea

    PubMed Central

    Hiromoto, Takeshi; Honjo, Eijiro; Noda, Naonobu; Tamada, Taro; Kazuma, Kohei; Suzuki, Masahiko; Blaber, Michael; Kuroki, Ryota

    2015-01-01

    UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3′- and 5′-hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5- and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments. PMID:25556637

  4. Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea.

    PubMed

    Hiromoto, Takeshi; Honjo, Eijiro; Noda, Naonobu; Tamada, Taro; Kazuma, Kohei; Suzuki, Masahiko; Blaber, Michael; Kuroki, Ryota

    2015-03-01

    UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3'- and 5'-hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1-O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5- and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments. PMID:25556637

  5. Using nonfluorescent Förster resonance energy transfer acceptors in protein binding studies.

    PubMed

    Ruan, Qiaoqiao; Skinner, Joseph P; Tetin, Sergey Y

    2009-10-15

    The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5-13(C10A) and full-length BNP1-32 with the dissociation constants 0.26+/-0.01 and 0.05+/-0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs=1.9 and 3.8) of the nonfluorescent FRET acceptor, K(D) values of 0.04+/-0.02 and 0.059(-0.004)(+0.006) nM, respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded K(D) values of 1.26+/-0.04, 1.25+/-0.05, and 1.14+/-0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies. PMID:19563765

  6. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-12-31

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  7. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-01-01

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  8. Microbial Diversity in Coastal Subsurface Sediments: a Cultivation Approach Using Various Electron Acceptors and Substrate Gradients

    PubMed Central

    Köpke, Beate; Wilms, Reinhard; Engelen, Bert; Cypionka, Heribert; Sass, Henrik

    2005-01-01

    Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, “Bacteroidetes,” “Fusobacteria,” Actinobacteria, and “Firmicutes.” Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors. PMID:16332756

  9. A Microplate Format Assay for Real-Time Screening for New Aldolases that Accept Aryl-Substituted Acceptor Substrates.

    PubMed

    Ma, Huan; Enugala, Thilak Reddy; Widersten, Mikael

    2015-12-01

    Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format. PMID:26449620

  10. Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180.

    PubMed

    Meng, Xiangfeng; Pijning, Tjaard; Dobruchowska, Justyna M; Gerwig, Gerrit J; Dijkhuizen, Lubbert

    2015-12-11

    α-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-ΔN from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite +1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (α1 → 6) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans. PMID:26507662

  11. Cooperative substrate binding by a diguanylate cyclase.

    PubMed

    Oliveira, Maycon C; Teixeira, Raphael D; Andrade, Maxuel O; Pinheiro, Glaucia M S; Ramos, Carlos H I; Farah, Chuck S

    2015-01-30

    XAC0610, from Xanthomonas citri subsp. citri, is a large multi-domain protein containing one GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domain, four PAS (Per-Arnt-Sim) domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity that leads to the production of cyclic di-GMP (c-di-GMP), a ubiquitous bacterial signaling molecule. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. Furthermore, experimental and in silico analyses suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately 5× greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo. PMID:25463434

  12. Investigation on the Synthesis of Shigella flexneri Specific Oligosaccharides Using Disaccharides as Potential Transglucosylase Acceptor Substrates.

    PubMed

    Salamone, Stéphane; Guerreiro, Catherine; Cambon, Emmanuelle; Hargreaves, Jason M; Tarrat, Nathalie; Remaud-Siméon, Magali; André, Isabelle; Mulard, Laurence A

    2015-11-20

    Chemo-enzymatic strategies hold great potential for the development of stereo- and regioselective syntheses of structurally defined bioactive oligosaccharides. Herein, we illustrate the potential of the appropriate combination of a planned chemo-enzymatic pathway and an engineered biocatalyst for the multistep synthesis of an important decasaccharide for vaccine development. We report the stepwise investigation, which led to an efficient chemical conversion of allyl α-d-glucopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside, the product of site-specific enzymatic α-d-glucosylation of a lightly protected non-natural disaccharide acceptor, into a pentasaccharide building block suitable for chain elongation at both ends. Successful differentiation between hydroxyl groups features the selective acylation of primary alcohols and acetalation of a cis-vicinal diol, followed by a controlled per-O-benzylation step. Moreover, we describe the successful use of the pentasaccharide intermediate in the [5 + 5] synthesis of an aminoethyl aglycon-equipped decasaccharide, corresponding to a dimer of the basic repeating unit from the O-specific polysaccharide of Shigella flexneri 2a, a major cause of bacillary dysentery. Four analogues of the disaccharide acceptor were synthesized and evaluated to reach a larger repertoire of O-glucosylation patterns encountered among S. flexneri type-specific polysaccharides. New insights on the potential and limitations of planned chemo-enzymatic pathways in oligosaccharide synthesis are provided. PMID:26340432

  13. The influence of substrate and electron acceptor availability on bioactive zone dynamics in porous media

    NASA Astrophysics Data System (ADS)

    Yolcubal, Irfan; Dorn, Jonathan G.; Maier, Raina M.; Brusseau, Mark L.

    2003-11-01

    Two approaches were used to investigate the influence of dissolved oxygen (DO) and substrate availability on the formation and dynamics of "bioactive zones" in a water-saturated porous medium. A bioactive zone is defined as a region where a microbial community is sufficiently active to metabolize bioavailable substrates. In the first approach, microbial activity was characterized by monitoring the spatial and temporal variability of DO and aqueous substrate (salicylate and naphthalene) concentrations during miscible-displacement experiments. In the second approach, microbial activity was monitored using multiple fiber optics emplaced in the porous medium to detect luminescence produced by Pseudomonas putida RB1353, a bioluminescent reporter organism that produces light when salicylate (an intermediate of naphthalene degradation) is present. The results of both approaches show that the location and size of the bioactive zones were influenced by in situ DO and substrate availability. When DO was not a limiting factor (i.e., lower substrate input concentrations), the bioactive zone encompassed the entire column, with the majority of the microbial activity occurring between the inlet and midpoint. However, as the availability of DO became limiting for the higher substrate input experiments, the size of the bioactive zone shrank and was ultimately limited to the proximity of the column inlet.

  14. Fresh look at electron-transfer mechanisms via the donor/acceptor bindings in the critical encounter complex.

    PubMed

    Rosokha, Sergiy V; Kochi, Jay K

    2008-05-01

    Seminal insights provided by the iconic R. S. Mulliken and his "charge-transfer" theory, H. Taube and his "outer/inner-sphere" mechanisms, R. A. Marcus and his "two-state non-adiabatic" theory, and N. S. Hush and his "intervalence" theory are each separately woven into the rich panoramic tapestry constituting chemical research into electron-transfer dynamics, and its mechanistic dominance for the past half century and more. In this Account, we illustrate how the simultaneous melding of all four key concepts allows sharp focus on the charge-transfer character of the critical encounter complex to evoke the latent facet of traditional electron-transfer mechanisms. To this end, we exploit the intervalence (electronic) transition that invariably accompanies the diffusive encounter of electron-rich organic donors (D) with electron-poor acceptors (A) as the experimental harbinger of the collision complex, which is then actually isolated and X-ray crystallographically established as loosely bound pi-stacked pairs of various aromatic and olefinic donor/acceptor dyads with uniform interplanar separations of r(DA) = 3.1 +/- 0.2 A. These X-ray structures, together with the spectral measurements of their intervalence transitions, lead to the pair of important electron-transfer parameters, H(DA) (electronic coupling element) versus lambdaT (reorganization energy), the ratio of which generally defines the odd-electron mobility within such an encounter complex in terms of the resonance stabilization of the donor/acceptor assembly [D, A] as opposed to the reorganization-energy penalty required for its interconversion to the electron-transfer state [D(+*), A(-*)]. We recognize the resonance-stabilization energy relative to the intrinsic activation barrier as the mechanistic binding factor, Q = 2H(DA)/lambdaT, to represent the quantitative measure of the highly variable continuum of inner-sphere/outer-sphere interactions that are possible within various types of precursor complexes

  15. Structural basis of substrate binding in WsaF, a rhamnosyltransferase from Geobacillus stearothermophilus.

    PubMed

    Steiner, Kerstin; Hagelueken, Gregor; Messner, Paul; Schäffer, Christina; Naismith, James H

    2010-03-26

    Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by alpha-1,3-, alpha-1,2-, and beta-1,2-linkages. The formation of the beta-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-beta-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-beta-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition. PMID:20097205

  16. Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases.

    PubMed

    Li, Yanhong; Xue, Mengyang; Sheng, Xue; Yu, Hai; Zeng, Jie; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Wang, Peng G; Chen, Xi

    2016-04-15

    β1-3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1-4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1-3Galβ1-4GlcβProN3 and Galβ1-3Galβ1-4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents. PMID:26968649

  17. Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

    PubMed Central

    Lee, Chi Chung; Fay, Aaron W.; Weng, Tsu-Chien; Krest, Courtney M.; Hedman, Britt; Hodgson, Keith O.; Hu, Yilin; Ribbe, Markus W.

    2015-01-01

    Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction. PMID:26515097

  18. The binding of substrate analogs to phosphotriesterase.

    PubMed

    Benning, M M; Hong, S B; Raushel, F M; Holden, H M

    2000-09-29

    Phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the detoxification of organophosphates such as the widely utilized insecticide paraoxon and the chemical warfare agent sarin. The three-dimensional structure of the enzyme is known from high resolution x-ray crystallographic analyses. Each subunit of the homodimer folds into a so-called TIM barrel, with eight strands of parallel beta-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the beta-barrel. Here, we describe the three-dimensional structure of PTE complexed with the inhibitor diisopropyl methyl phosphonate, which serves as a mimic for sarin. Additionally, the structure of the enzyme complexed with triethyl phosphate is also presented. In the case of the PTE-diisopropyl methyl phosphonate complex, the phosphoryl oxygen of the inhibitor coordinates to the more solvent-exposed zinc ion (2.5 A), thereby lending support to the presumed catalytic mechanism involving metal coordination of the substrate. In the PTE-triethyl phosphate complex, the phosphoryl oxygen of the inhibitor is positioned at 3.4 A from the more solvent-exposed zinc ion. The two structures described in this report provide additional molecular understanding for the ability of this remarkable enzyme to hydrolyze such a wide range of organophosphorus substrates. PMID:10871616

  19. Structure-guided redesign of D-fructose-6-phosphate aldolase from E. coli: remarkable activity and selectivity towards acceptor substrates by two-point mutation.

    PubMed

    Gutierrez, Mariana; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Clapés, Pere

    2011-05-28

    Structure-guided re-design of the acceptor binding site of D-fructose-6-phosphate aldolase from E. coli leads to the construction of FSA A129S/A165G double mutant with an activity between 5- to >900-fold higher than that of wild-type towards N-Cbz-aminoaldehyde derivatives. PMID:21499643

  20. Substrate binding accelerates the conformational transitions and substrate dissociation in multidrug efflux transporter AcrB

    PubMed Central

    Wang, Beibei; Weng, Jingwei; Wang, Wenning

    2015-01-01

    The tripartite efflux pump assembly AcrAB-TolC is the major multidrug resistance transporter in E. coli. The inner membrane transporter AcrB is a homotrimer, energized by the proton movement down the transmembrane electrochemical gradient. The asymmetric crystal structures of AcrB with three monomers in distinct conformational states [access (A), binding (B) and extrusion (E)] support a functional rotating mechanism, in which each monomer of AcrB cycles among the three states in a concerted way. However, the relationship between the conformational changes during functional rotation and drug translocation has not been totally understood. Here, we explored the conformational changes of the AcrB homotrimer during the ABE to BEA transition in different substrate-binding states using targeted MD simulations. It was found that the dissociation of substrate from the distal binding pocket of B monomer is closely related to the concerted conformational changes in the translocation pathway, especially the side chain reorientation of Phe628 and Tyr327. A second substrate binding at the proximal binding pocket of A monomer evidently accelerates the conformational transitions as well as substrate dissociation in B monomer. The acceleration effect of the multi-substrate binding mode provides a molecular explanation for the positive cooperativity observed in the kinetic studies of substrate efflux and deepens our understanding of the functional rotating mechanism of AcrB. PMID:25918513

  1. Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase.

    PubMed

    Salinas, S R; Petruk, A A; Brukman, N G; Bianco, M I; Jacobs, M; Marti, M A; Ielpi, L

    2016-06-01

    GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site. PMID:27099353

  2. Central action of dendrotoxin: selective reduction of a transient K conductance in hippocampus and binding to localized acceptors

    SciTech Connect

    Halliwell, J.V.; Othman, I.B.; Pelchen-Matthews, A.; Dolly, J.O.

    1986-01-01

    Dendrotoxin, a small single-chain protein from the venom of Dendroaspis angusticeps, is highly toxic following intracerebroventricular injection into rats. Voltage-clamp analysis of CA/sub 1/ neurons in hippocampal slices, treated with tetrodotoxin, revealed that nanomolar concentrations of dendrotoxin reduce selectively a transient, voltage-dependent K conductance. Epileptiform activity known to be induced by dendrotoxin can be attributed to such an action. Membrane currents not affected directly by the toxin include (i) Ca-activated K conductance; (ii) noninactivating voltage-dependent K conductance; (iii) inactivating the noninactivating Ca conductances; (iv) persistent inward (anomalous) rectifier current. Persistence of the effects of the toxin when Cd was included to suppress spontaneous transmitter release indicates a direct action on the neuronal membrane. Using biologically active, /sup 125/I-labeled dendrotoxin, protein acceptor sites of high affinity were detected on cerebrocortical synapotosomal membranes and sections of rat brain. In hippocampus, toxin binding was shown autoradiographically to reside in synapse-rich and white matter regions, with lower levels in cell body layers. This acceptor is implicated in the action of toxin because its affinities for dendrotoxin congeners are proportional to their central neurotoxicities and potencies in reducing the transient, voltage-dependent K conductance.

  3. Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

    DOE PAGESBeta

    Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin; Xu, Jian-He

    2015-02-25

    A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase r

  4. Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

    SciTech Connect

    Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin; Xu, Jian-He

    2015-02-25

    A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of the substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase r

  5. Structural Basis of Fatty Acid Substrate Binding to Cyclooxygenase-2*

    PubMed Central

    Vecchio, Alex J.; Simmons, Danielle M.; Malkowski, Michael G.

    2010-01-01

    The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H2 from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co3+-protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 Å, respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted in AA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates. PMID:20463020

  6. Oxygen as Acceptor.

    PubMed

    Borisov, Vitaliy B; Verkhovsky, Michael I

    2015-01-01

    Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate-specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophosphate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and dimethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O₂ is served by two major oxidoreductases (oxidases), cytochrome bo₃ encoded by cyoABCDE and cytochrome bd encoded by cydABX. Terminal oxidases of aerobic respiratory chains of bacteria, which use O₂ as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo₃ and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo₃ and cytochrome bd. The E. coli membrane contains three types of quinones that all have an octaprenyl side chain (C₄₀). It has been proposed that the bo₃ oxidase can have two ubiquinone-binding sites with different affinities. "WHAT'S NEW" IN THE REVISED ARTICLE: The revised article comprises additional information about subunit composition of cytochrome bd and its role in bacterial resistance to nitrosative and oxidative stresses. Also, we present the novel data on the electrogenic function of appBCX-encoded cytochrome bd-II, a second bd-type oxidase that had been thought not to contribute to generation of a proton motive force in E

  7. An additional substrate binding site in a bacterial phenylalanine hydroxylase

    PubMed Central

    Ronau, Judith A.; Paul, Lake N.; Fuchs, Julian E.; Corn, Isaac R.; Wagner, Kyle T.; Liedl, Klaus R.; Abu-Omar, Mahdi M.; Das, Chittaranjan

    2014-01-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

  8. Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase

    SciTech Connect

    Gruber, Todd D.; Borrok, M. Jack; Westler, William M.; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5{prime}-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 {angstrom} resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the

  9. Dendrotoxin acceptor from bovine synaptic plasma membranes. Binding properties, purification and subunit composition of a putative constituent of certain voltage-activated K+ channels.

    PubMed Central

    Parcej, D N; Dolly, J O

    1989-01-01

    Dendrotoxin is a snake polypeptide that blocks selectively and potently certain voltage-sensitive, fast-activating K+ channels in the nervous system, where it binds with high affinity to membranous acceptors. Herein, the acceptor protein for dendrotoxin in bovine synaptic membranes is solubilized in active form and its complete purification achieved by affinity chromatography, involving a novel elution procedure. This putative K+-channel constituent is shown to be a large oligomeric glycoprotein containing two major subunits, with Mr values of 75,000 and 37,000. Images Fig. 2. PMID:2930493

  10. In-silico analysis of binding site features and substrate selectivity in plant flavonoid-3-O glycosyltransferases (F3GT) through molecular modeling, docking and dynamics simulation studies.

    PubMed

    Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C G

    2014-01-01

    Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

  11. In-Silico Analysis of Binding Site Features and Substrate Selectivity in Plant Flavonoid-3-O Glycosyltransferases (F3GT) through Molecular Modeling, Docking and Dynamics Simulation Studies

    PubMed Central

    Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C.G.

    2014-01-01

    Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

  12. Prenylated Rab acceptor 1 (PRA1) inhibits TCF/{beta}-catenin signaling by binding to {beta}-catenin

    SciTech Connect

    Kim, Jong-Tae; Cho, Mi-Young; Choi, Seung-Chul; Kim, Jung Woo; Chae, Suhn-Kee; Yoon, Do-Young; Kim, Jae Wha . E-mail: wjkim@kribb.re.kr; Lim, Jong-Seok . E-mail: jslim@sookmyung.ac.kr

    2006-10-13

    The prenylated Rab acceptor 1 (PRA1) is a ubiquitously expressed 21 kDa protein containing two transmembrane domains that possibly induce its localization to the Golgi complex. It binds to prenylated Rab GTPases and VAMP2. In this study, we report that PRA1-overexpressing cells exhibited a significantly retarded growth rate as compared to that of the mock-transfected cells, and the transcriptional activity of TCF, as evaluated by TOPflash luciferase reporter assay, was profoundly reduced in the PRA1-overexpressed cells. These intracellular functions of PRA1 were verified by introducing deletion mutant or site-directed mutants, or small interfering RNA of PRA1. In addition, the translocation of {beta}-catenin from the cytosol to the nucleus was blocked to a significant degree in the PRA1-cells, and the interaction of PRA1 and {beta}-catenin was identified by confocal microscopy and immunoprecipitation analysis. Finally, we observed that the inhibition of TCF/{beta}-catenin signaling by PRA1 is associated with ERK1/2 dephosphorylation. Therefore, our data suggest that the in vivo modulation of PRA1 may be involved in TCF/{beta}-catenin signaling, as well as cellular proliferation and tumorigenesis.

  13. Structural basis of substrate discrimination and integrin binding by autotaxin

    SciTech Connect

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna J.S.; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig W.; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

  14. Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

    SciTech Connect

    Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao; Xue, Fei; Wu, Zhixin; Shen, Wei; Li, Jun; Zhou, Zhe; Ding, Yi; Zhao, Qi; Zhang, Xuejun C.; Liao, Ming; Bartlam, Mark; Rao, Zihe

    2008-07-21

    Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) MP{sup pro} and a severe acute respiratory syndrome CoV (SARS-CoV) M{sup pro} mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M{sup pro}. A monomeric form of IBV M{sup pro} was identified for the first time in CoV M{sup pro} structures. A comparison of these two structures to other available M{sup pro} structures provides new insights for the design of substrate-based inhibitors targeting CoV M{sup pro}s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M{sup pro} and was found to demonstrate in vitro inactivation of IBV M{sup pro} and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M{sup pro}.

  15. ABC transporters: one, two or four extracytoplasmic substrate-binding sites?

    PubMed Central

    van der Heide, Tiemen; Poolman, Bert

    2002-01-01

    Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C-terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator. PMID:12370206

  16. UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

    PubMed Central

    Kilani, A F; Liu, F

    1999-01-01

    RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes. PMID:10496224

  17. Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

    PubMed Central

    Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P.; Abramson, Jeff; Quick, Matthias; Shi, Lei

    2015-01-01

    The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

  18. Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H+:Cl− exchanger

    PubMed Central

    Picollo, Alessandra; Xu, Yanyan; Johner, Niklaus; Bernèche, Simon; Accardi, Alessio

    2012-01-01

    Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies suggested that all exchangers share two fundamental properties: substrate binding is antagonistic and coupling is maintained by preventing shuttling of the empty transporter. The CLC Cl−: H+ exchangers control the homeostasis of cellular compartments in most living organisms but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a critical glutamate. The simultaneous binding of H+ and Cl− gives rise to a fully-loaded state incompatible with conventional mechanisms. Mutations in the Cl− transport pathway identically alter the stoichiometries of Cl−: H+ exchange and binding. We propose that the thermodynamics of synergistic substrate binding determine the stoichiometry of transport rather than the kinetics of conformational changes and ion binding. PMID:22484316

  19. Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

    NASA Astrophysics Data System (ADS)

    Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

    2016-03-01

    Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

  20. Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding

    PubMed Central

    Schuman, Brock; Persson, Mattias; Landry, Roxanne C.; Polakowski, Robert; Weadge, Joel T.; Seto, Nina O. L.; Borisova, Svetlana N.; Palcic, Monica M.; Evans, Stephen V.

    2011-01-01

    A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177–195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog. PMID:20655926

  1. Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

    PubMed Central

    Singh, Appu Kumar; Mondal, Alok K.; Kumaran, S.

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism. PMID:23049810

  2. Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter

    PubMed Central

    De Paepe, Anne; Vanakker, Olivier M.

    2014-01-01

    The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein (ABCC6), primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS) and leukotriene C4 (LTC4) were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3) was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE. PMID:25062064

  3. Coupling Substrate and Ion Binding to Extracellular Gate of a Sodium-Dependent Aspartate Transporter

    SciTech Connect

    Boudker,O.; Ryan, R.; Yernool, D.; Shimamoto, K.; Gouaux, E.

    2007-01-01

    Secondary transporters are integral membrane proteins that catalyze the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt{sub Ph}, a sodium (Na{sup +})-coupled aspartate transporter, defining sites for aspartate, two sodium ions and D,L-threo-{beta}-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound {alpha}-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.

  4. The role of amino acid electron-donor/acceptor atoms in host-cell binding peptides is associated with their 3D structure and HLA-binding capacity in sterile malarial immunity induction

    SciTech Connect

    Patarroyo, Manuel E.; Almonacid, Hannia; Moreno-Vranich, Armando

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Fundamental residues located in some HABPs are associated with their 3D structure. Black-Right-Pointing-Pointer Electron-donor atoms present in {beta}-turn, random, distorted {alpha}-helix structures. Black-Right-Pointing-Pointer Electron-donor atoms bound to HLA-DR53. Black-Right-Pointing-Pointer Electron-acceptor atoms present in regular {alpha}-helix structure bound to HLA-DR52. -- Abstract: Plasmodium falciparum malaria continues being one of the parasitic diseases causing the highest worldwide mortality due to the parasite's multiple evasion mechanisms, such as immunological silence. Membrane and organelle proteins are used during invasion for interactions mediated by high binding ability peptides (HABPs); these have amino acids which establish hydrogen bonds between them in some of their critical binding residues. Immunisation assays in the Aotus model using HABPs whose critical residues had been modified have revealed a conformational change thereby enabling a protection-inducing response. This has improved fitting within HLA-DR{beta}1{sup Asterisk-Operator} molecules where amino acid electron-donor atoms present in {beta}-turn, random or distorted {alpha}-helix structures preferentially bound to HLA-DR53 molecules, whilst HABPs having amino acid electron-acceptor atoms present in regular {alpha}-helix structure bound to HLA-DR52. This data has great implications for vaccine development.

  5. Solid-binding Proteins for Modification of Inorganic Substrates

    NASA Astrophysics Data System (ADS)

    Coyle, Brandon Laurence

    Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. In this work, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of various proteins to exploit their binding ability. In addition, we conducted a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbon and silicon surfaces. Through these insights, we were able to develop proteins suitable for dispersing graphene flakes and carbon nanotubes in aqueous solutions, while retaining protein activity. Additionally, our investigation into the mechanisms of adhesion for our carbon binding peptides inspired a cheap, disposable protein purification system that is more than 10x cheaper than commonly used His-tag protein purification. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

  6. Structural Analysis of Substrate and Effector Binding in Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

    SciTech Connect

    Dey, Sanghamitra; Burton, Rodney L.; Grant, Gregory A.; Sacchettini, James C.

    2008-08-20

    The crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase has been solved with bound effector, L-serine, and substrate, hydroxypyruvic acid phosphate, at resolutions of 2.7 and 2.4 {angstrom}, respectively. The subunits display the same extreme asymmetry as seen in the apo-structure and provide insight into the mode of serine binding and closure of the active site. Mutagenesis studies confirm the identity of the main residues involved in serine binding and suggest that the poly glycine stretch in the loop that contains the locus for the 160{sup o} rotation that leads to subunit asymmetry may have a larger role in folding than in catalysis. The lack of electron density for the cofactor, NADH, in any of the crystals examined led us to study binding by stopped flow kinetic analysis. The kinetic data suggest that productive NADH binding, that would support catalytic turnover, is dependent on the presence of substrate. This observation, along with the binding of substrate in the active site, but in an unproductive conformation, suggests a possible mechanism where initial binding of substrate leads to enhanced interaction with cofactor accompanied by a rearrangement of catalytically critical residue side chains. Furthermore, comparison to the structure of a truncated form of human D-3-phosphoglycerate dehydrogenase with cofactor and a substrate analog, provides insight into the conformational changes that occur during catalysis.

  7. Deconstructing the DGAT1 Enzyme: Membrane Interactions at Substrate Binding Sites

    PubMed Central

    Lopes, Jose L. S.; Beltramini, Leila M.; Wallace, Bonnie A.; Araujo, Ana P. U.

    2015-01-01

    Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates. PMID:25719207

  8. Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.

    PubMed

    Gao, Dahai; Chundawat, Shishir P S; Sethi, Anurag; Balan, Venkatesh; Gnanakaran, S; Dale, Bruce E

    2013-07-01

    Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme-substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph I(β) to III(I) results in 40-50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings. PMID:23784776

  9. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

    PubMed Central

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšík, Martin; Peclinovská, Lucie; Majer, Pavel; Strisovsky, Kvido

    2014-01-01

    The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG. PMID:25216680

  10. Substrate binding properties of potato tuber ADP-glucose pyrophosphorylase as determined by isothermal titration calorimetry.

    PubMed

    Cakir, Bilal; Tuncel, Aytug; Green, Abigail R; Koper, Kaan; Hwang, Seon-Kap; Okita, Thomas W; Kang, ChulHee

    2015-06-01

    Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism. PMID:25953126

  11. Substrate Binding and Catalytic Mechanism of Human Choline Acetyltransferase

    SciTech Connect

    Kim,A.; Rylett, J.; Shilton, B.

    2006-01-01

    Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 {angstrom} along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the K{sub M} for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.

  12. Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter*

    PubMed Central

    Bippes, Christian A.; Zeltina, Antra; Casagrande, Fabio; Ratera, Merce; Palacin, Manuel; Muller, Daniel J.; Fotiadis, Dimitrios

    2009-01-01

    We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state. PMID:19419962

  13. Progress on binding CO{sub 2} in mineral substrates

    SciTech Connect

    Lackner, K.S.; Butt, D.P.; Wendt, C.H.

    1996-10-01

    Based on current estimates of reserves, coal could satisfy even a very much increased world energy demand for centuries, if only the emission of CO{sub 2} disposal that is based on combining CO{sub 2} chemically with abundant raw materials to form stable carbonate minerals. A major advantage of this method is that the resulting waste product is thermodynamically stable and environmentally neutral. It is therefore possible to store large quantities permanently with minimal environmental impact and without the danger of an accidental release of CO{sub 2} which has proven fatal in quantities far smaller than contemplated here. The raw materials to bind CO{sub 2} exist in nature in large quantities. They are readily accessible and far exceed what would be required to bind all CO{sub 2} that could possibly be generated by burning the entire fossil fuel reserves. In this paper the authors outline a specific process that they are currently investigating. The initial rough cost estimate of about 3{cents}/kWh is encouraging. The availability of a CO{sub 2} fixation technology would serve as insurance in case global warming, or the perception of global warming, would cause severe restrictions on CO{sub 2} emissions. If the increased energy demand of a growing world population is to be satisfied from coal, the implementation of such a technology would be unavoidable.

  14. Identifying Sequential Substrate Binding at the Single-Molecule Level by Enzyme Mechanical Stabilization

    PubMed Central

    Rivas-Pardo, Jaime Andrés; Alegre-Cebollada, Jorge; Ramírez-Sarmiento, César A.; Fernandez, Julio M.; Guixé, Victoria

    2015-01-01

    Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate I, which is stabilized in the presence of Mg·ADP-, the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate-1*. Finally, in the presence of both substrates, the unfolding force of intermediates-1 and -1*, increases as a consequence of the domain closure. These results show that SMFS could be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology. PMID:25840594

  15. Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes*

    PubMed Central

    Martinez, Jacobo; Truffault, Vincent; Hothorn, Michael

    2015-01-01

    Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family. PMID:26221030

  16. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  17. DNA sequencing using polymerase substrate-binding kinetics.

    PubMed

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  18. Freeze-drying of enzymes in case of water-binding and non-water-binding substrates.

    PubMed

    Pisano, Roberto; Rasetto, Valeria; Barresi, Antonello A; Kuntz, Florent; Aoude-Werner, Dalal; Rey, Louis

    2013-11-01

    Enzymes typically have a critical instability, which dominates both formulation and process development. In this paper, the ability to preserve the enzyme activity during freeze-drying was investigated for both water-binding and non-water-binding substrates. For this purpose, acid phosphatase was used as model protein. In addition, a procedure for the fast development of a freeze-drying cycle is shown. For the secondary drying part, the effect of processing temperature and time on enzyme activity was investigated. The enzyme activity decreased continuously during secondary drying, with a dramatic drop associated with processing temperatures higher than 293 K. Besides product temperature, the residual moisture level and water mobility are also important. As the residual moisture level and water mobility depend on the product formulation, the stabilizing effect against the enzyme deactivation was studied for a number of formulations which contain different additives, that is, sucrose, lactose, mannitol, and poly-vinylpyrrolidone, with a dramatic activity loss associated with crystallizing excipients. This study also confirmed that not all water-binding substrates can successfully protect the enzyme against deactivation. In fact, water-binding substrates containing reducing sugars (lactose) showed the highest loss of activity. PMID:23500114

  19. Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase.

    PubMed

    Møller, Marie S; Windahl, Michael S; Sim, Lyann; Bøjstrup, Marie; Abou Hachem, Maher; Hindsgaul, Ole; Palcic, Monica; Svensson, Birte; Henriksen, Anette

    2015-03-27

    Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis. PMID:25562209

  20. Mechanism of Substrate and Inhibitor Binding of Rhodobacter Capsulatus Xanthine Dehydrogenase

    SciTech Connect

    Dietzel, U.; Kuper, J; Doebbler, J; Schulte, A; Truglio, J; Leimkuhler, S; Kisker, C

    2009-01-01

    Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (ae)2 heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (EB232Q) to prevent substrate turnover. The hypoxanthine- and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward ArgB-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a Ki of 103.57 {+-} 18.96 nm for pterin-6-aldehyde.

  1. Characterization of substrate binding of the WW domains in human WWP2 protein.

    PubMed

    Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

    2015-07-01

    WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. PMID:25999310

  2. X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

    SciTech Connect

    Shishova,E.; Yu, F.; Miller, D.; Faraldos, J.; Zhao, Y.; Coates, R.; Allemann, R.; Cane, D.; Christianson, D.

    2008-01-01

    The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg2+-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PPi) coproduct. The 2.1- Angstroms resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PPi and Mg2+B to monomer D. The 1.89- Angstroms resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg2+Baccompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4- Angstroms resolution structure of the complex with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the open conformation and the binding of PPi, Mg2+B, and Mg2+C to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent 'snapshots' of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.

  3. Computational method for relative binding energies of enzyme-substrate complexes.

    PubMed

    Zhang, T; Koshland, D E

    1996-02-01

    A computational method for estimating the relative binding free energies of enzyme-substrate complexes is described that combines electrostatic and solvation models and X-ray crystallographic data. The polar contribution is evaluated by the Poisson-Boltzman equation. The nonpolar contribution is evaluated by solvent transfer data and surface area calculations. This algorithm was used to calculate the relative binding energies of 63 pairs of nine different mutant proteins with seven different substituted R-malate substrates of Escherichia coli isocitrate dehydrogenase. Comparison of calculated values with the experimentally observed values shows a high degree of correlation. PMID:8745413

  4. Selectivity of substrate binding and ionization of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

    PubMed

    Luanloet, Thikumporn; Sucharitakul, Jeerus; Chaiyen, Pimchai

    2015-08-01

    2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the

  5. Insight into determinants of substrate binding and transport in a multidrug efflux protein

    PubMed Central

    Alegre, Kamela O.; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J.

    2016-01-01

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that ‘aromatic clamps’ formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such ‘clamps’ may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins. PMID:26961153

  6. Insight into determinants of substrate binding and transport in a multidrug efflux protein.

    PubMed

    Alegre, Kamela O; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J

    2016-01-01

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins. PMID:26961153

  7. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites

    PubMed Central

    Ragusa, Michael J.; Dancheck, Barbara; Critton, David A.; Nairn, Angus C.; Page, Rebecca; Peti, Wolfgang

    2010-01-01

    The serine/threonine Protein Phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with ≥200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1’s specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form and binds PP1, through a folding-upon-binding mechanism, in an elongated fashion, blocking one of PP1’s three putative substrate binding sites, without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1’s activity toward a model substrate in vitro, without affecting its ability to dephosphorylate its neuronal substrate GluR1. Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity. PMID:20305656

  8. Structural insights into the substrate-binding mechanism for a novel chitosanase.

    PubMed

    Lyu, Qianqian; Wang, Song; Xu, Wenhua; Han, Baoqin; Liu, Wanshun; Jones, David N M; Liu, Weizhi

    2014-07-15

    Chitosanase is able to specifically cleave β-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion. PMID:24766439

  9. Tentative Identification of the Second Substrate Binding Site in Arabidopsis Phytochelatin Synthase

    PubMed Central

    Chia, Ju-Chen; Yang, Chien-Chih; Sui, Yu-Ting; Lin, Shin-Yu; Juang, Rong-Huay

    2013-01-01

    Phytochelatin synthase (PCS) uses the substrates glutathione (GSH, γGlu-Cys-Gly) and a cadmium (Cd)-bound GSH (Cd∙GS2) to produce the shortest phytochelatin product (PC2, (γGlu-Cys)2-Gly) through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12–218) by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS) as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation) resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W) for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed. PMID:24340051

  10. Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates*

    PubMed Central

    Gao, Feng; Kight, Alicia D.; Henderson, Rory; Jayanthi, Srinivas; Patel, Parth; Murchison, Marissa; Sharma, Priyanka; Goforth, Robyn L.; Kumar, Thallapuranam Krishnaswamy Suresh; Henry, Ralph L.; Heyes, Colin D.

    2015-01-01

    Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP. PMID:25918165

  11. Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2.

    PubMed

    Poulin, Myles B; Schneck, Jessica L; Matico, Rosalie E; Hou, Wangfang; McDevitt, Patrick J; Holbert, Marc; Schramm, Vern L

    2016-06-01

    Nuclear receptor-binding SET domain protein 2 (NSD2) is a histone H3 lysine 36 (H3K36)-specific methyltransferase enzyme that is overexpressed in a number of cancers, including multiple myeloma. NSD2 binds to S-adenosyl-l-methionine (SAM) and nucleosome substrates to catalyze the transfer of a methyl group from SAM to the ε-amino group of histone H3K36. Equilibrium binding isotope effects and density functional theory calculations indicate that the SAM methyl group is sterically constrained in complex with NSD2, and that this steric constraint is released upon nucleosome binding. Together, these results show that nucleosome binding to NSD2 induces a significant change in the chemical environment of enzyme-bound SAM. PMID:27183271

  12. Insights into Substrate and Metal Binding from the Crystal Structure of Cyanobacterial Aldehyde Deformylating Oxygenase with Substrate Bound

    PubMed Central

    2015-01-01

    The nonheme diiron enzyme cyanobacterial aldehyde deformylating oxygenase, cADO, catalyzes the highly unusual deformylation of aliphatic aldehydes to alkanes and formate. We have determined crystal structures for the enzyme with a long-chain water-soluble aldehyde and medium-chain carboxylic acid bound to the active site. These structures delineate a hydrophobic channel that connects the solvent with the deeply buried active site and reveal a mode of substrate binding that is different from previously determined structures with long-chain fatty acids bound. The structures also identify a water channel leading to the active site that could facilitate the entry of protons required in the reaction. NMR studies examining 1-[13C]-octanal binding to cADO indicate that the enzyme binds the aldehyde form rather than the hydrated form. Lastly, the fortuitous cocrystallization of the metal-free form of the protein with aldehyde bound has revealed protein conformation changes that are involved in binding iron. PMID:25222710

  13. Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein.

    PubMed

    Ferreira, Ricardo J; Ferreira, Maria-José U; dos Santos, Daniel J V A

    2013-07-22

    P-Glycoprotein (Pgp) is one of the best characterized ABC transporters, often involved in the multidrug-resistance phenotype overexpressed by several cancer cell lines. Experimental studies contributed to important knowledge concerning substrate polyspecificity, efflux mechanism, and drug-binding sites. This information is, however, scattered through different perspectives, not existing a unifying model for the knowledge available for this transporter. Using a previously refined structure of murine Pgp, three putative drug-binding sites were hereby characterized by means of molecular docking. The modulator site (M-site) is characterized by cross interactions between both Pgp halves herein defined for the first time, having an important role in impairing conformational changes leading to substrate efflux. Two other binding sites, located next to the inner leaflet of the lipid bilayer, were identified as the substrate-binding H and R sites by matching docking and experimental results. A new classification model with the ability to discriminate substrates from modulators is also proposed, integrating a vast number of theoretical and experimental data. PMID:23802684

  14. Discovery of a sensitive, selective, and tight binding fluorogenic substrate of bovine plasma amine oxidase

    PubMed Central

    Ling, Ke-Qing; Sayre, Lawrence M.

    2008-01-01

    We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (Km 183±14 nM), and yet is metabolized fairly quickly (kcat 0.690±0.010 s−1), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorometric non-coupled assay that is two orders of magnitude more sensitive than the spectrophotometric benzylamine assay. The discovery of ANETA involved elaboration of the lead compound 6-methoxy-2-naphthalenemethaneamine by structure-based design, which recognized the ancillary cation binding site of BPAO as the most significant structural features controlling binding affinity. Structure-based design further ensured a high level of selectivity: ANETA is a good substrate of BPAO, but is not a substrate of either porcine kidney diamine oxidase (pkDAO) or rat liver monoamine oxidase (MAO-B). ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond directly to the enzyme activity in real time. PMID:19053593

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

    PubMed Central

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

    2009-01-01

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

  16. Enhancement of binding kinetics on affinity substrates by laser point heating induced transport.

    PubMed

    Wang, Bu; Cheng, Xuanhong

    2016-03-01

    Enhancing the time response and detection limit of affinity-binding based biosensors is an area of active research. For diffusion limited reactions, introducing active mass transport is an effective strategy to reduce the equilibration time and improve surface binding. Here, a laser is focused on the ceiling of a microchamber to generate point heating, which introduces natural advection and thermophoresis to promote mass transport to the reactive floor. We first used the COMSOL simulation to study how the kinetics of ligand binding is influenced by the optothermal effect. Afterwards, binding of biotinylated nanoparticles to NeutrAvidin-treated substrates is quantitatively measured with and without laser heating. It is discovered that laser induced point heating reduces the reaction half-life locally, and the reduction improves with the natural advection velocity. In addition, non-uniform ligand binding on the substrate is induced by the laser with predictable binding patterns. This optothermal strategy holds promise to improve the time-response and sensitivity of biosensors and microarrays. PMID:26898559

  17. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-04-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  18. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

    PubMed

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ. PMID:27112153

  19. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  20. Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.

    PubMed

    Taabazuing, Cornelius Y; Fermann, Justin; Garman, Scott; Knapp, Michael J

    2016-01-19

    The Fe(2+)/α-ketoglutarate (αKG)-dependent oxygenases use molecular oxygen to conduct a wide variety of reactions with important biological implications, such as DNA base excision repair, histone demethylation, and the cellular hypoxia response. These enzymes follow a sequential mechanism in which O2 binds and reacts after the primary substrate binds, making those structural factors that promote productive O2 binding central to their chemistry. A large challenge in this field is to identify strategies that engender productive turnover. Factor inhibiting HIF (FIH) is a Fe(2+)/αKG-dependent oxygenase that forms part of the O2 sensing machinery in human cells by hydroxylating the C-terminal transactivation domain (CTAD) found within the HIF-1α protein. The structure of FIH was determined with the O2 analogue NO bound to Fe, offering the first direct insight into the gas binding geometry in this enzyme. Through a combination of density functional theory calculations, {FeNO}(7) electron paramagnetic resonance spectroscopy, and ultraviolet-visible absorption spectroscopy, we demonstrate that CTAD binding stimulates O2 reactivity by altering the orientation of the bound gas molecule. Although unliganded FIH binds NO with moderate affinity, the bound gas can adopt either of two orientations with similar stability; upon CTAD binding, NO adopts a single preferred orientation that is appropriate for supporting oxidative decarboxylation. Combined with other studies of related enzymes, our data suggest that substrate-induced reorientation of bound O2 is the mechanism utilized by the αKG oxygenases to tightly couple O2 activation to substrate hydroxylation. PMID:26727884

  1. Structural analysis reveals the substrate-binding mechanism for the expanded substrate specificity of mutant meso-diaminopimelate dehydrogenase.

    PubMed

    Liu, Weidong; Guo, Rey-Ting; Chen, Xi; Li, Zhe; Gao, Xiuzhen; Huang, Chun-Hsiang; Wu, Qiaqing; Feng, Jinhui; Zhu, Dunming

    2015-04-13

    A meso-diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D-amino acids other than its native substrate. Site-directed mutagenesis similar to that carried out on the residues mutated by Vedha-Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D-amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso-diaminopimelate (meso-DAP), D-leucine (D-leu), and 4-methyl-2-oxopentanoic acid (MOPA) were solved. meso-DAP was found in an area outside the catalytic cavity; this suggested a possible two-step substrate-binding mechanism for meso-DAP. D-leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso-diaminopimelate dehydrogenases. PMID:25754803

  2. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin.

    PubMed

    Karpowich, Nathan K; Song, Jinmei; Wang, Da-Neng

    2016-07-31

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters. PMID:27312125

  3. Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase.

    PubMed

    Ingram-Smith, Cheryl; Woods, Barrett I; Smith, Kerry S

    2006-09-26

    AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site. PMID:16981708

  4. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

    PubMed Central

    Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

    2016-01-01

    Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

  5. Binding and Channeling of Alternative Substrates in the Enzyme DmpFG: a Molecular Dynamics Study

    PubMed Central

    Smith, Natalie E.; Vrielink, Alice; Attwood, Paul V.; Corry, Ben

    2014-01-01

    DmpFG is a bifunctional enzyme comprised of an aldolase subunit, DmpG, and a dehydrogenase subunit, DmpF. The aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. In this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, DmpG, using molecular dynamics. All substrates investigated are easily accommodated within the active site, binding with free energy values comparable to the physiological substrate 4-hydroxy-2-ketovalerate. Subsequently, umbrella sampling was utilized to obtain free energy surfaces for the aldehyde intermediates (which would be generated from the aldolase reaction on each of these substrates) to move through the channel to the dehydrogenase DmpF. Small substrates were channeled with limited barriers in an energetically feasible process. We show that the barriers preventing bulky intermediates such as benzaldehyde from moving through the wild-type protein can be removed by selective mutation of channel-lining residues, demonstrating the potential for tailoring this enzyme to allow its use for the synthesis of specific chemical products. Furthermore, positions of transient escape routes in this flexible channel were determined. PMID:24739167

  6. Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding

    PubMed Central

    Eytan, Esther; Moshe, Yakir; Braunstein, Ilana; Hershko, Avram

    2006-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G1 phase of the cell cycle. The activity of APC/C in mitosis and in G1 requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C–Cdc20 contain a recognition motif called the “destruction box” (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C–activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation we examined this problem by an “isotope-trapping” approach that directly demonstrates productive substrate binding. With this method we found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays we found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. Our results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding. PMID:16455800

  7. Substrate recognition and specificity of double-stranded RNA binding proteins.

    PubMed

    Vuković, Lela; Koh, Hye Ran; Myong, Sua; Schulten, Klaus

    2014-06-01

    Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA-RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2'-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA-RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA-RNA hybrids. Our study reveals the structural and molecular basis of protein-RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins. PMID:24801449

  8. HC[triple bond]P and H3C-C[triple bond]P as proton acceptors in protonated complexes containing two phosphorus bases: structures, binding energies, and spin-spin coupling constants.

    PubMed

    Alkorta, Ibon; Elguero, José; Bene, Janet E Del

    2007-10-01

    Ab initio calculations at the MP2/aug'-cc-pVTZ level have been carried out to investigate the structures and binding energies of cationic complexes involving protonated sp, sp2, and sp3 phosphorus bases as proton donor ions and the sp-hybridized phosphorus bases H-C[triple bond]P and H3C-C[triple bond]P as proton acceptors. These proton-bound complexes exhibit a variety of structural motifs, but all are stabilized by interactions that occur through the pi cloud of the acceptor base. The binding energies of these complexes range from 6 to 15 kcal/mol. Corresponding complexes with H3C-C[triple bond]P as the proton acceptor are more stable than those with H-C[triple bond]P as the acceptor, a reflection of the greater basicity of H3C-C[triple bond]P. In most complexes with sp2- or sp3-hybridized P-H donor ions, the P-H bond lengthens and the P-H stretching frequency is red-shifted relative to the corresponding monomers. Complex formation also leads to a lengthening of the C[triple bond]P bond and a red shift of the C[triple bond]P stretching vibration. The two-bond coupling constants 2pihJ(P-P) and 2pihJ(P-C) are significantly smaller than 2hJ(P-P) and 2hJ(P-C) for complexes in which hydrogen bonding occurs through lone pairs of electrons on P or C. This reflects the absence of significant s electron density in the hydrogen-bonding regions of these pi complexes. PMID:17760429

  9. Structural domains in NADPH: Protochlorophyllide oxidoreductases involved in catalysis and substrate binding. Final report

    SciTech Connect

    Timko, Michael P.

    1999-09-24

    Until recently little direct information was available about specific structural determinants within the light-dependent NADPH: protochlorophyllide oxidoreductases (PORs) required for substrate and cofactor binding, catalytic activity, and thylakoid membrane localization. Based on our previous DOE-funded studies, during the past year we brought to fruition a number of ongoing experiments, initiated several new avenues of investigations, and overall have made considerable progress towards establishing the basic structural parameters governing POR function. Our studies to date have defined residues and domains involved in substrate and cofactor binding and catalysis, and elaborated on the mechanism for membrane localization of POR in developing plastids. Our results and their significance, as well as our work in progress, are detailed.

  10. Carboxy-Terminus Recruitment Induced by Substrate Binding in Eukaryotic Fructose Bis-phosphate Aldolases

    SciTech Connect

    Lafrance-Vanasse,J.; Sygusch, J.

    2007-01-01

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 {angstrom} resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P{sub 1}-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P{sub 1}-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P{sub 1}-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine.

  11. Carboxy-terminus recruitment induced by substrate binding in eukaryotic fructose bis-phosphate aldolases.

    PubMed

    Lafrance-Vanasse, Julien; Sygusch, Jurgen

    2007-08-21

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 A resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P1-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P1-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P1-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine. PMID:17661446

  12. Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.

    PubMed

    Tarry, Michael J; Schäfer, Eva; Chen, Shuyun; Buchanan, Grant; Greene, Nicholas P; Lea, Susan M; Palmer, Tracy; Saibil, Helen R; Berks, Ben C

    2009-08-11

    The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein. PMID:19666509

  13. Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase

    PubMed Central

    Shehata, Saifeldin N.; Hunter, Roger W.; Ohta, Eriko; Peggie, Mark W.; Lou, Hua Jane; Sicheri, Frank; Zeqiraj, Elton; Turk, Benjamin E.; Sakamoto, Kei

    2012-01-01

    PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+ 1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at + 4, but not at + 3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide > 100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase. PMID:22796189

  14. Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects.

    PubMed

    Roston, Daniel; Cui, Qiang

    2016-09-14

    Enzymes are powerful catalysts, and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction's transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around-as with classic "induced fit" models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step. PMID:27541005

  15. Binding of mouse immunoglobulin G to polylysine-coated glass substrate for immunodiagnosis

    NASA Astrophysics Data System (ADS)

    Vashist, Sandeep Kumar; Tewari, Rupinder; Bajpai, Ram Prakash; Bharadwaj, Lalit Mohan; Raiteri, Roberto

    2006-12-01

    We report a method for immobilizing mouse immunoglobulin G (IgG) on polylysine-coated glass substrate for immunodiagnostic applications. Mouse IgG molecules were immobilized on polylysine-coated glass substrate employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and protein A. The amino groups of the polylysine-coated glass slide were cross linked to the carboxyl groups of protein A employing EDC crosslinker. Protein A was employed as it binds to the constant Fc region of antibodies keeping their antigen binding sites on the variable F ab region free to bind to antigens. The qualitative analysis of surface immobilized mouse IgG was done by fluorescent microscopy employing fluorescein isothiocyanate (FITC) labeled mouse IgG molecules. The immobilization densities of protein A and mouse IgG were determined by 3, 3', 4, 4'-tetramethyl benzidine (TMB) substrate assay employing horse radish peroxidise labelled molecules and were found to be 130 +/- 17 ng/cm2 and 596 +/- 31 ng/cm2 respectively. The biomolecular coatings analyzed by atomic force microscopy (AFM) were found to be uniform.

  16. Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

    PubMed

    Jamaluddin, M Fairuz B; Bailey, Ulla-Maja; Schulz, Benjamin L

    2014-12-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  17. Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation*

    PubMed Central

    Jamaluddin, M. Fairuz B.; Bailey, Ulla-Maja; Schulz, Benjamin L.

    2014-01-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  18. Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.

    PubMed Central

    Chase, T

    1986-01-01

    Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger. PMID:3545182

  19. The Use of Nucleosome Substrates Improves Binding of SAM Analogs to SETD8.

    PubMed

    Strelow, John M; Xiao, Min; Cavitt, Rachel N; Fite, Nathan C; Margolis, Brandon J; Park, Kyu-Jin

    2016-09-01

    SETD8 is the methyltransferase responsible for monomethylation of lysine at position 20 of the N-terminus of histone H4 (H4K20). This activity has been implicated in both DNA damage and cell cycle progression. Existing biochemical assays have utilized truncated enzymes containing the SET domain of SETD8 and peptide substrates. In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used. PMID:27369108

  20. Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase

    SciTech Connect

    Warren, M.J.; Jordan, P.M.

    1988-12-13

    The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA/sup -/ mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free ..cap alpha..-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino(5-/sup 14/C)levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the ..cap alpha..-substituted substrate analogue ..cap alpha..-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cummulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH/sub 3/ or H/sub 2/O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

  1. Understanding the molecular basis of substrate binding specificity of PTB domains

    PubMed Central

    Sain, Neetu; Tiwari, Garima; Mohanty, Debasisa

    2016-01-01

    Protein-protein interactions mediated by phosphotyrosine binding (PTB) domains play a crucial role in various cellular processes. In order to understand the structural basis of substrate recognition by PTB domains, multiple explicit solvent atomistic simulations of 100ns duration have been carried out on 6 PTB-peptide complexes with known binding affinities. MM/PBSA binding energy values calculated from these MD trajectories and residue based statistical pair potential score show good correlation with the experimental dissociation constants. Our analysis also shows that the modeled structures of PTB domains can be used to develop less compute intensive residue level statistical pair potential based approaches for predicting interaction partners of PTB domains. PMID:27526776

  2. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination. PMID:26244568

  3. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGESBeta

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  4. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    SciTech Connect

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  5. The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

    PubMed Central

    Mitić, Nataša; Hadler, Kieran S.; Gahan, Lawrence R; Hengge, Alvan C.; Schenk, Gerhard

    2010-01-01

    The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe3+-Fe2+ pig PAP (uteroferrin; Uf) and its Fe3+-Mn2+ derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe2+ by Mn2+ lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe3+ site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a μ-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe3+ site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe3+. The faster rates of interaction with the Fe3+ site in the Fe3+-Mn2+ derivative than the native Fe3+-Fe2+ form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme. PMID:20433174

  6. Conformational Changes in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase upon Substrate Binding

    PubMed Central

    Baños-Sanz, José Ignacio; Sanz-Aparicio, Julia; Whitfield, Hayley; Hamilton, Chris; Brearley, Charles A.; González, Beatriz

    2012-01-01

    Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) catalyzes the synthesis of inositol 1,2,3,4,5,6-hexakisphosphate from ATP and IP5. Inositol 1,2,3,4,5,6-hexakisphosphate is implicated in crucial processes such as mRNA export, DNA editing, and phosphorus storage in plants. We previously solved the first structure of an IP5 2-K, which shed light on aspects of substrate recognition. However, failure of IP5 2-K to crystallize in the absence of inositide prompted us to study putative conformational changes upon substrate binding. We have made mutations to residues on a region of the protein that produces a clasp over the active site. A W129A mutant allowed us to capture IP5 2-K in its different conformations by crystallography. Thus, the IP5 2-K apo-form structure displays an open conformation, whereas the nucleotide-bound form shows a half-closed conformation, in contrast to the inositide-bound form obtained previously in a closed conformation. Both nucleotide and inositide binding produce large conformational changes that can be understood as two rigid domain movements, although local changes were also observed. Changes in intrinsic fluorescence upon nucleotide and inositide binding are in agreement with the crystallographic findings. Our work suggests that the clasp might be involved in enzyme kinetics, with the N-terminal lobe being essential for inositide binding and subsequent conformational changes. We also show how IP5 2-K discriminates between inositol 1,3,4,5-tetrakisphosphate and 3,4,5,6-tetrakisphosphate enantiomers and that substrate preference can be manipulated by Arg130 mutation. Altogether, these results provide a framework for rational design of specific inhibitors with potential applications as biological tools for in vivo studies, which could assist in the identification of novel roles for IP5 2-K in mammals. PMID:22745128

  7. Structural Basis for Substrate Binding and the Catalytic Mechanism of Type III Pantothenate Kinase

    SciTech Connect

    Yang, Kun; Strauss, Erick; Huerta, Carlos; Zhang, Hong

    2008-07-15

    Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high K{sub m} for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the 'acetate and sugar kinase/heat shock protein 70/actin' (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.

  8. Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters

    PubMed Central

    Yu, You; Zhou, Mingze; Kirsch, Franziska; Xu, Congqiao; Zhang, Li; Wang, Yu; Jiang, Zheng; Wang, Na; Li, Jun; Eitinger, Thomas; Yang, Maojun

    2014-01-01

    The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu2+- and Ni2+-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co2+. Indeed, the structure of TtNikM2 containing a bound Co2+ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters. PMID:24366337

  9. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.

    PubMed Central

    Trifillis, P; Day, N; Kiledjian, M

    1999-01-01

    Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions. PMID:10445881

  10. Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A.

    PubMed

    Li, Yongchao; Irwin, Diana C; Wilson, David B

    2007-05-01

    Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(-1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(-2) to Glc(-4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(-2) to Glc(-4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate. PMID:17369336

  11. The role of propionates in substrate binding to heme oxygenase from Neisseria meningitidis; A NMR study†

    PubMed Central

    Peng, Dungeng; Ma, Li-Hua; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

    2012-01-01

    Heme oxygenase, HO, cleaves hemin into biliverdin, iron and CO. For mammalian HOs, both native hemin propionates are required for substrate binding and activity. The HO from the pathogenic bacterium Neisseria meningitidis, NmHO, possesses a crystallographically undetected C-terminal fragment that by solution 1H NMR is found to fold and interact with the active site. One of the substrate propionates has been proposed to form a salt bridge to the C-terminus rather than to the conventional buried cationic side chain in other HOs. Moreover, the C-terminal dipeptide Arg208His209 cleaves spontaneously over ~24 hours at a rate dependent on substituent size. 2D 1H NMR of NmHO azide complexes with hemins with selectively deleted or rearranged propionates all bind to NmHO with a structurally conserved active site as reflected in optical spectra and NMR NOESY cross peak and hyperfine shift patterns. In contrast to mammalian HOs, NmHO requires only a single propionate interacting with the buried terminus of Lys16 to exhibit full activity and tolerates the existence of a propionate at the exposed 8-position. The structure of the C-terminus is qualitatively retained upon deletion of the 7-propionate but a dramatic change in the 7-propionate carboxylate 13C chemical shift upon C-terminal cleavage confirms its role in the interaction with the C-terminus. The stronger hydrophobic contacts between pyrroles A and B with NmHO contribute more substantially to the substrate binding free energy than in mammalian HOs, “liberating” one propionate to stabilize the C-terminus. The functional implications of the C-terminus in product release are discussed. PMID:22913621

  12. Reductive dehalogenation by cytochrome P450CAM: Substrate binding and catalysis

    SciTech Connect

    Li, S.; Wackett, L.P. )

    1993-09-14

    Biological reductive dehalogenation reactions are important in environmental detoxification of organohalides. Only scarce information is available on the enzymology underlying these reactions. Cytochrome P450CAM with a known X-ray structure and well-studied oxygenase reaction cycle, has been studied for its ability to reduce carbon-halogen bonds under anaerobic conditions. The reductive reactions functioned with NADH and the physiological electron-transfer proteins or by using artificial electron donors to reduce cytochrome P450CAM. Halogenated methane and ethane substrates were transformed by a two-electron reduction and subsequent protonation, beta-elimination, or alpha-elimination to yield alkanes, alkene, or carbene-derived products, respectively. Halogenated substrates bound to the camphor binding site as indicated by saturable changes in the Fe(III)-heme spin state upon substrate addition. Hexachloromethane was bound with a dissociation constant (KD) of 0.7 microM and caused > 95% shift from low- to high-spin iron. Ethanes bearing fewer chlorine substituents were bound with increasing dissociation constants and gave lesser degrees of iron spin-state change. Camphor competitively inhibited hexachloroethane reduction with an inhibitor constant (KI) similar to the dissociation constant for camphor (KI = KD = 0.9 microM). Rate determinations with pentachloroethane indicated a 100-fold higher enzyme V/K compared to the second-order rate constant for hematin free in solution. These studies on substrate binding and catalysis will help reveal how biological systems enzymatically reduce carbon-halogen bonds in the environment.

  13. Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions.

    PubMed

    Brooks, S P; Storey, K B

    1993-05-12

    The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell. PMID:8350861

  14. Substrate Binding Mode and its Implication on Drug Design for Botulinum Neurotoxin A

    SciTech Connect

    Kumaran, D.; Rawat, R; Ahmed, A; Swaminathan, S

    2008-01-01

    The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide 197QRATKM202 and its variant 197RRATKM202 to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5? sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1?-Arg198, occupies the S1? site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2? subsite is formed by Arg363, Asn368 and Asp370, while S3? subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4?-Lys201 makes hydrogen bond with Gln162. P5?-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

  15. Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

    PubMed

    Onesti, S; Desogus, G; Brevet, A; Chen, J; Plateau, P; Blanquet, S; Brick, P

    2000-10-24

    Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding. PMID:11041850

  16. Squeezing at entrance of proton transport pathway in proton-translocating pyrophosphatase upon substrate binding.

    PubMed

    Huang, Yun-Tzu; Liu, Tseng-Huang; Lin, Shih-Ming; Chen, Yen-Wei; Pan, Yih-Jiuan; Lee, Ching-Hung; Sun, Yuh-Ju; Tseng, Fan-Gang; Pan, Rong-Long

    2013-07-01

    Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding. PMID:23720778

  17. Substrate Binding Promotes Formation of the Skp1-Cul1-Fbxl3 (SCFFbxl3) Protein Complex*

    PubMed Central

    Yumimoto, Kanae; Muneoka, Tetsuya; Tsuboi, Tomohiro; Nakayama, Keiichi I.

    2013-01-01

    The Skp1–Cul1–F-box protein (SCF) complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the complex being regulated in part at the level of complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCFFbxl3 complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCFFbxl3 complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCFFbxl3. In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity. PMID:24085301

  18. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases

    PubMed Central

    2015-01-01

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design. PMID:26709959

  19. Enzyme-Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer.

    PubMed

    Schelvis, Johannes P M; Zhu, Xuling; Gindt, Yvonne M

    2015-10-13

    Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 10(6) M(-1) is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼10(4) M(-1) s(-1) and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 10(5) M(-1) s(-1) and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search. PMID:26393415

  20. 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.

  1. Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

    SciTech Connect

    Ku,S.; Yip, P.; Cornell, K.; Riscoe, M.; Behr, J.; Guillerm, G.; Howell, P.

    2007-01-01

    The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp{sup 250}-Glu{sup 252}). In addition, the glycine-rich loop of the protein, analogous to the 'Gly triad' in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp{sup 233} of the catalytic HGD motif, a novel twin arginine motif (Arg{sup 340}/Arg{sup 341}), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

  2. Mechanistic Insights from the Binding of Substrate and Carbocation Intermediate Analogues to Aristolochene Synthase

    PubMed Central

    Chen, Mengbin; Al-lami, Naeemah; Janvier, Marine; D'Antonio, Edward L.; Faraldos, Juan A.; Cane, David E.; Allemann, Rudolf K.; Christianson, David W.

    2013-01-01

    Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg2+ ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg2+ ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg2+3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism. PMID:23905850

  3. Associative Memory Acceptors.

    ERIC Educational Resources Information Center

    Card, Roger

    The properties of an associative memory are examined in this paper from the viewpoint of automata theory. A device called an associative memory acceptor is studied under real-time operation. The family "L" of languages accepted by real-time associative memory acceptors is shown to properly contain the family of languages accepted by one-tape,…

  4. A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase*

    PubMed Central

    Lietzan, Adam D.; St. Maurice, Martin

    2013-01-01

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes. PMID:23698000

  5. Calculation of substrate binding affinities for a bacterial GH78 rhamnosidase through molecular dynamics simulations

    PubMed Central

    Grandits, Melanie; Michlmayr, Herbert; Sygmund, Christoph; Oostenbrink, Chris

    2013-01-01

    Ram2 from Pediococcus acidilactici is a rhamnosidase from the glycoside hydrolase family 78. It shows remarkable selectivity for rutinose rather than para-nitrophenyl-alpha-l-rhamnopyranoside (p-NPR). Molecular dynamics simulations were performed using a homology model of this enzyme, in complex with both substrates. Free energy calculations lead to predicted binding affinities of −34.4 and −30.6 kJ mol−1 respectively, agreeing well with an experimentally estimated relative free energy of 5.4 kJ mol−1. Further, the most relevant binding poses could be determined. While p-NPR preferably orients its rhamnose moiety toward the active site, rutinose interacts most strongly with its glucose moiety. A detailed hydrogen bond analysis confirms previously implicated residues in the active site (Asp217, Asp222, Trp226, Asp229 and Glu488) and quantifies the importance of individual residues for the binding. The most important amino acids are Asp229 and Phe339 which are involved in many interactions during the simulations. While Phe339 was observed in more simulations, Asp229 was involved in more persistent interactions (forming an average of at least 2 hydrogen bonds during the simulation). These analyses directly suggest mutations that could be used in a further experimental characterization of the enzyme. This study shows once more the strength of computer simulations to rationalize and guide experiments at an atomic level. PMID:23914137

  6. Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes*

    PubMed Central

    Luechapanichkul, Rinrada; Chen, Xianwen; Taha, Hashem A.; Vyas, Shubham; Guan, Xiaoyan; Freitas, Michael A.; Hadad, Christopher M.; Pei, Dehua

    2013-01-01

    Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/φ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/φ)(T/S)(D/E)pY(G/A/S/Q) (where φ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X1–6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp164, which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo. PMID:23322772

  7. Structural modelling of substrate binding and inhibition in penicillin V acylase from Pectobacterium atrosepticum.

    PubMed

    Avinash, V S; Panigrahi, Priyabrata; Suresh, C G; Pundle, Archana V; Ramasamy, Sureshkumar

    2013-08-01

    Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation. PMID:23850621

  8. N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis.

    PubMed

    Hoskins, Aaron A; Morar, Mariya; Kappock, T Joseph; Mathews, Irimpan I; Zaugg, Judith B; Barder, Timothy E; Peng, Paul; Okamoto, Akimitsu; Ealick, Steven E; Stubbe, JoAnne

    2007-03-13

    N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR. PMID:17298082

  9. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

    SciTech Connect

    Reger,A.; Carney, J.; Gulick, A.

    2007-01-01

    The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.

  10. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d. PMID:26766115

  11. Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

    PubMed Central

    Fernando, Harshica; Rumfeldt, Jessica A. O.; Davydova, Nadezhda Y.; Halpert, James R.; Davydov, Dmitri R.

    2010-01-01

    1. The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W and L211F/D214E was studied with fluorescence resonance energy transfer (FRET) and absorbance spectroscopy. 2. Whereas in the wild type enzyme the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (nH) around 1.7 (S50 = 11.7 μM), the mutants showed no cooperativity (nH ≤ 1.1) with unchanged S50 values. 3. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high affinity interaction is characterized by a dissociation constant (KD) ≤ 1.0 μM, whereas the KD of the second binding has the same magnitude as the S50. 4. Theoretical analysis of a two-step binding model suggests that nH values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event. 5. Alteration of cooperativity in the mutants is caused by a partial displacement of the “spin-shifting” step. Whereas in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift upon binding of the first substrate molecule. PMID:21143007

  12. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T; Durell, Stewart R; Ambudkar, Suresh V

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each

  13. Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.

    PubMed

    Geronimo, Inacrist; Denning, Catherine A; Rogers, W Eric; Othman, Thaer; Huxford, Tom; Heidary, David K; Glazer, Edith C; Payne, Christina M

    2016-06-28

    Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability. PMID:27267136

  14. Autoproteolytic activation of ThnT results in structural reorganization necessary for substrate binding and catalysis

    PubMed Central

    Buller, Andrew R; Labonte, Jason W; Freeman, Michael F; Wright, Nathan T; Schildbach, Joel F; Townsend, Craig A

    2012-01-01

    cis-Autoproteolysis is a post-translational modification necessary for the function of ThnT, an enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. This modification generates an N-terminal threonine nucleophile that is used to hydrolyze the pantetheinyl moiety of its natural substrate. We determined the crystal structure of autoactivated ThnT to 1.8 Å through X-ray crystallography. Comparison to a mutationally inactivated precursor structure revealed several large conformational rearrangements near the active site. To probe the relevance of these transitions, we designed a pantetheine-like chloromethyl ketone (CMK) inactivator and co-crystallized it with ThnT. Although this class of inhibitor has been in use for several decades, the mode of inactivation had not been determined for an enzyme that uses an N-terminal nucleophile. The co-crystal structure revealed the CMK bound to the N-terminal nucleophile of ThnT through an ether linkage and analysis suggests inactivation through a direct displacement mechanism. More importantly, this inactivated complex shows three regions of ThnT that are critical to the formation of the substrate binding pocket undergo rearrangement upon autoproteolysis. Comparison of ThnT with other autoproteolytic enzymes of disparate evolutionary lineage revealed a high degree similarity within the pro-enzyme active site, reflecting shared chemical constraints. However, after autoproteolysis many enzymes, like ThnT, are observed to rearrange to accommodate their specific substrate. We propose this is a general phenomenon, whereby autoprocessing systems with shared chemistry may possess similar structural features that dissipate upon rearrangement into a mature state. PMID:22706025

  15. Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase.

    PubMed

    Johnson, Troy A; Mcleod, Matthew J; Holyoak, Todd

    2016-01-26

    Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Previous work has demonstrated that the enzyme cycles between a catalytically inactive open state and a catalytically active closed state. The transition of the enzyme between these states requires the transition of several active site loops to shift from mobile, disordered structural elements to stable ordered states. The mechanism by which these disorder-order transitions are coupled to the ligation state of the active site however is not fully understood. To further investigate the mechanisms by which the mobility of the active site loops is coupled to enzymatic function and the transitioning of the enzyme between the two conformational states, we have conducted structural and functional studies of point mutants of E89. E89 is a proposed key member of the interaction network of mobile elements as it resides in the R-loop region of the enzyme active site. These new data demonstrate the importance of the R-loop in coordinating interactions between substrates at the OAA/PEP binding site and the mobile R- and Ω-loop domains. In turn, the studies more generally demonstrate the mechanisms by which the intrinsic ligand binding energy can be utilized in catalysis to drive unfavorable conformational changes, changes that are subsequently required for both optimal catalytic activity and fidelity. PMID:26709450

  16. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    PubMed Central

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  17. Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm.

    PubMed

    Triboulet, Sébastien; Bougault, Catherine M; Laguri, Cédric; Hugonnet, Jean-Emmanuel; Arthur, Michel; Simorre, Jean-Pierre

    2015-10-01

    In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of β-lactam antibiotics is bypassed by L,D-transpeptidases that form unusual 3 → 3 peptidoglycan cross-links. β-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium L,D-transpeptidase Ldt(fm) by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt(fm) that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt(fm) active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β-lactams. PMID:26101813

  18. The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

    PubMed Central

    Ejendal, Karin F.K.; Diop, Ndeye Khady; Schweiger, Linda C.; Hrycyna, Christine A.

    2006-01-01

    Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding. PMID:16815914

  19. The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase.

    PubMed

    Genta, Fernando A; Dumont, Alexandra F; Marana, Sandro R; Terra, Walter R; Ferreira, Clélia

    2007-09-01

    Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity. PMID:17720633

  20. Substrate binding to cytochrome P450-2J2 in Nanodiscs detected by nanoplasmonic Lycurgus cup arrays.

    PubMed

    Plucinski, Lisa; Ranjan Gartia, Manas; Arnold, William R; Ameen, Abid; Chang, Te-Wei; Hsiao, Austin; Logan Liu, Gang; Das, Aditi

    2016-01-15

    Cytochrome P450s are the primary enzymes involved in phase I drug metabolism. They are an important target for early drug discovery research. However, high-throughput drug screening of P450s is limited by poor protein stability and lack of consistent measurement of binding events. Here we present the detection of substrate binding to cytochrome P450-2J2 (CYP2J2), the predominant P450 in the human heart, using a combination of Nanodisc technology and a nanohole plasmonic sensor called nanoplasmonic Lycurgus cup array (nanoLCA). The Nanodisc, a nanoscale membrane bilayer disc, is used to stabilize the protein on the metallic plasmonic surface. Absorption spectroscopy of seven different substrates binding to CYP2J2 in solution showed that they are all type I, resulting in shifting of the protein bands to lower wavelengths (blue shift). Detection on the nanoLCA sensor also showed spectral blue shifts of CYP2J2 following substrate binding. Finite Difference Time Domain (FDTD) electromagnetic simulation suggested that the blue shift on the nanoLCA is because of the hybridization of plasmon polariton Bloch wave and the electronic resonance of the heme group of CYP2J2. We found the plasmonic properties of the nanoLCA sensor to be highly reproducible, which allowed comparisons among the different substrates at different concentrations. Further, due to the unique spectral properties of the nanoLCA sensor, including the transmission of a single color, we were able to perform colorimetric detection of the binding events. These results indicate that a resonance plasmonic sensing mechanism can be used to distinguish between different substrates of the same binding type at different concentrations binding to P450s and that the nanoLCA sensor has the potential to provide consistent high-throughput measurements of this system. PMID:26334592

  1. Microfluidic Channels on Nanopatterned Substrates: Monitoring Protein Binding to Lipid Bilayers with Surface-Enhanced Raman Spectroscopy

    PubMed Central

    Banerjee, Amrita; Perez-Castillejos, R.; Hahn, D.; Smirnov, Alex I.; Grebel, H.

    2013-01-01

    We used Surface Enhanced Raman Spectroscopy (SERS) to detect binding events between streptavidin and biotinylated lipid bilayers. The binding events took place at the surface between microfluidic channels and anodized aluminum oxide (AAO) with the latter serving as substrates. The bilayers were incorporated in the substrate pores. It was revealed that non-bound molecules were easily washed away and that large suspended cells (Salmonella enterica) are less likely to interfere with the monitoring process: when focusing to the lower surface of the channel, one may resolve mostly the bound molecules. PMID:24932024

  2. NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions.

    PubMed

    Aachmann, Finn L; Sørlie, Morten; Skjåk-Bræk, Gudmund; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

    2012-11-13

    Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu(2+) (K(d) = 55 nM; from isothermal titration calorimetry) and higher preference for Cu(1+) (K(d) ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu(2+) of 275 mV using a thermodynamic cycle). Strong binding of Cu(1+) was also reflected in a reduction in the pK(a) values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a (2)H/(1)H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates. PMID:23112164

  3. NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

    PubMed Central

    Aachmann, Finn L.; Sørlie, Morten; Skjåk-Bræk, Gudmund; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2012-01-01

    Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu2+ (Kd = 55 nM; from isothermal titration calorimetry) and higher preference for Cu1+ (Kd ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu2+ of 275 mV using a thermodynamic cycle). Strong binding of Cu1+ was also reflected in a reduction in the pKa values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a 2H/1H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates. PMID:23112164

  4. The primary substrate binding site in the b' domain of ERp57 is adapted for endoplasmic reticulum lectin association.

    PubMed

    Russell, Sarah J; Ruddock, Lloyd W; Salo, Kirsi E H; Oliver, Jason D; Roebuck, Quentin P; Llewellyn, David H; Roderick, H Llewelyn; Koivunen, Peppi; Myllyharju, Johanna; High, Stephen

    2004-04-30

    ERp57 is a member of the protein disulfide isomerase (PDI) family that is located in the endoplasmic reticulum (ER) and characterized by its specificity for glycoproteins. Substrate selection by ERp57 is dependent upon its formation of discrete complexes with two ER resident lectins, soluble calreticulin and membrane-bound calnexin. It is these two lectins that directly associate with glycoproteins bearing correctly trimmed oligosaccharide side chains. Thus, ERp57 is presented with a preselected set of substrates upon which it can act, and the specific binding of calreticulin and calnexin to ERp57 is pivotal to the functions of the resulting complexes. To gain further insights into the formation of these ERp57-ER lectin complexes, we have investigated the regions of ERp57 that are specifically required for its binding to calreticulin. Using a quantitative pull-down assay to investigate the binding of ERp57/PDI chimeras to calreticulin, we define the b and b' domains of ERp57 as the minimal elements that are sufficient for complex formation. This analysis further identifies a novel role for the distinctive C-terminal extension of ERp57 in reconstituting complex formation to wild type levels. Using our understanding of substrate binding to the b' domain of PDI as a paradigm, we show that alterations to specific residues in the b' domain of ERp57 dramatically reduce or completely abolish its binding to calreticulin. On the basis of these data, we propose a model where the region of ERp57 equivalent to the primary substrate binding site of archetypal PDI is occupied by calreticulin and suggest that the ER lectins act as adaptor molecules that define the substrate specificity of ERp57. PMID:14871899

  5. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    SciTech Connect

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  6. Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations.

    PubMed

    Elmabrouk, Zainab H; Vincent, Florence; Zhang, Meng; Smith, Nicola L; Turkenburg, Johan P; Charnock, Simon J; Black, Gary W; Taylor, Edward J

    2011-03-01

    Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of β-sheets, the β-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and β-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases. PMID:21287626

  7. Self-assembled nanospheres with multiple endohedral binding sites pre-organize catalysts and substrates for highly efficient reactions.

    PubMed

    Wang, Qi-Qiang; Gonell, Sergio; Leenders, Stefan H A M; Dürr, Maximilian; Ivanović-Burmazović, Ivana; Reek, Joost N H

    2016-03-01

    Tuning reagent and catalyst concentrations is crucial in the development of efficient catalytic transformations. In enzyme-catalysed reactions the substrate is bound-often by multiple non-covalent interactions-in a well-defined pocket close to the active site of the enzyme; this pre-organization facilitates highly efficient transformations. Here we report an artificial system that co-encapsulates multiple catalysts and substrates within the confined space defined by an M12L24 nanosphere that contains 24 endohedral guanidinium-binding sites. Cooperative binding means that sulfonate guests are bound much more strongly than carboxylates. This difference has been used to fix gold-based catalysts firmly, with the remaining binding sites left to pre-organize substrates. This strategy was applied to a Au(I)-catalysed cyclization of acetylenic acid to enol lactone in which the pre-organization resulted in much higher reaction rates. We also found that the encapsulated sulfonate-containing Au(I) catalysts did not convert neutral (acid) substrates, and so could have potential in the development of substrate-selective catalysis and base-triggered on/off switching of catalysis. PMID:26892553

  8. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.

    PubMed

    Fischer, Marcus; Hopkins, Adam P; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G; Hubbard, Roderick E; Thomas, Gavin H

    2015-11-01

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

  9. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

    PubMed Central

    Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

    2015-01-01

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

  10. Self-assembled nanospheres with multiple endohedral binding sites pre-organize catalysts and substrates for highly efficient reactions

    NASA Astrophysics Data System (ADS)

    Wang, Qi-Qiang; Gonell, Sergio; Leenders, Stefan H. A. M.; Dürr, Maximilian; Ivanović-Burmazović, Ivana; Reek, Joost N. H.

    2016-03-01

    Tuning reagent and catalyst concentrations is crucial in the development of efficient catalytic transformations. In enzyme-catalysed reactions the substrate is bound—often by multiple non-covalent interactions—in a well-defined pocket close to the active site of the enzyme; this pre-organization facilitates highly efficient transformations. Here we report an artificial system that co-encapsulates multiple catalysts and substrates within the confined space defined by an M12L24 nanosphere that contains 24 endohedral guanidinium-binding sites. Cooperative binding means that sulfonate guests are bound much more strongly than carboxylates. This difference has been used to fix gold-based catalysts firmly, with the remaining binding sites left to pre-organize substrates. This strategy was applied to a Au(I)-catalysed cyclization of acetylenic acid to enol lactone in which the pre-organization resulted in much higher reaction rates. We also found that the encapsulated sulfonate-containing Au(I) catalysts did not convert neutral (acid) substrates, and so could have potential in the development of substrate-selective catalysis and base-triggered on/off switching of catalysis.

  11. Binding of polychlorinated biphenyls to aquatic humic substances: The role of substrate and sorbate properties on partitioning

    SciTech Connect

    Uhle, M.E.; Chin, Y.P.; Aiken, G.R.; McKnight, D.M.

    1999-08-15

    Two ortho- (2,2{prime},5 and 2,2{prime}5,6{prime}) and a non-ortho- (3,3{prime},4,4{prime}) substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho-substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1{prime} carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate`s higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.

  12. Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis.

    PubMed

    Cookson, Tammie V M; Castell, Alina; Bulloch, Esther M M; Evans, Genevieve L; Short, Francesca L; Baker, Edward N; Lott, J Shaun; Parker, Emily J

    2014-07-01

    AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5'-phosphoribosyl-1'-pyrophosphate) to anthranilate to form PRA (5'-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP. PMID:24712732

  13. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins.

    PubMed

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-04-19

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  14. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    PubMed Central

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  15. Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

    PubMed

    Banerjee, Rupa; Jayaraj, Gopal Gunanathan; Peter, Joshua Jebakumar; Kumar, Vignesh; Mapa, Koyeli

    2016-08-01

    DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures. PMID:27248857

  16. Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates

    PubMed Central

    2013-01-01

    Introduction Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. Method We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. Results Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. Conclusion Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film. PMID:23819686

  17. Binding of polychlorinated biphenyls to aquatic humic substances: The role of substrate and sorbate properties on partitioning

    USGS Publications Warehouse

    Uhle, M.E.; Chin, Y.-P.; Aiken, G.R.; McKnight, Diane M.

    1999-01-01

    Two ortho- (2,2',5 and 2,2',5,6') and a non-ortho- (3,3',4,4') substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho- substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1' carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.Two ortho- (2,2???,5 and 2,2???,5,6???) and a non-ortho- (3,3???,4,4???) substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho-substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1??? carbon bond, thereby making the molecule less able to interact

  18. Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

    PubMed

    Pradhan, Anupam; Tripathi, Abhai K; Desai, Prashant V; Mukherjee, Prasenjit K; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2009-01-01

    The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate

  19. Alternansucrase acceptor products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The regioselectivity of alternansucrase (EC 2.4.1.140) differs from dextransucrase (EC 2.4.1.5) in ways that can be useful for the synthesis of novel oligosaccharide structures. For example, it has been recently shown that the major oligosaccharides produced when maltose is the acceptor include one...

  20. Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

    PubMed Central

    Aprile, Francesco A.; Dhulesia, Anne; Stengel, Florian; Roodveldt, Cintia; Benesch, Justin L. P.; Tortora, Paolo; Robinson, Carol V.; Salvatella, Xavier; Dobson, Christopher M.; Cremades, Nunilo

    2013-01-01

    Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. PMID:23840795

  1. Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

    PubMed Central

    Choudhury, Manabendra D.; Modi, Mahendra K.

    2014-01-01

    ADP-glucose pyrophosphorylase (AGPase) is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide. PMID:25276800

  2. Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase

    PubMed Central

    2016-01-01

    mediate the formation of Mn(III) for catalysis upon substrate binding. PMID:27014926

  3. Three holes bound to a double acceptor - Be(+) in germanium

    NASA Technical Reports Server (NTRS)

    Haller, E. E.; Mcmurray, R. E., Jr.; Falicov, L. M.; Haegel, N. M.; Hansen, W. L.

    1983-01-01

    A double acceptor binding three holes has been observed for the first time with photoconductive far-infrared spectroscopy in beryllium-doped germanium single crystals. This new center, Be(+), has a hole binding energy of about 5 meV and is only present when free holes are generated by ionization of either neutral shallow acceptors or neutral Be double acceptors. The Be(+) center thermally ionizes above 4 K. It disappears at a uniaxial stress higher than about a billion dyn/sq cm parallel to (111) as a result of the lifting of the valence-band degeneracy.

  4. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  5. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  6. Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

    PubMed Central

    Romano, Keith P.; Ali, Akbar; Royer, William E.; Schiffer, Celia A.

    2010-01-01

    Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates. PMID:21084633

  7. Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context

    SciTech Connect

    Wang, Hui; Elferich, Johannes; Gouaux, Eric

    2012-02-13

    Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-{beta}-D-glucopyranoside ({beta}-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT-substrate complexes in lipid bicelles and in {beta}-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

  8. Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate

    SciTech Connect

    Pazy, Y.; Motaleb, M.A.; Guarnieri, M.T.; Charon, N.W.; Zhao, R.; Silversmith, R.E.

    2010-04-05

    Two-component signal transduction systems are widespread in prokaryotes and control numerous cellular processes. Extensive investigation of sensor kinase and response regulator proteins from many two-component systems has established conserved sequence, structural, and mechanistic features within each family. In contrast, the phosphatases which catalyze hydrolysis of the response regulator phosphoryl group to terminate signal transduction are poorly understood. Here we present structural and functional characterization of a representative of the CheC/CheX/FliY phosphatase family. The X-ray crystal structure of Borrelia burgdorferi CheX complexed with its CheY3 substrate and the phosphoryl analogue BeF{sub 3}{sup -} reveals a binding orientation between a response regulator and an auxiliary protein different from that shared by every previously characterized example. The surface of CheY3 containing the phosphoryl group interacts directly with a long helix of CheX which bears the conserved (E - X{sub 2} - N) motif. Conserved CheX residues Glu96 and Asn99, separated by a single helical turn, insert into the CheY3 active site. Structural and functional data indicate that CheX Asn99 and CheY3 Thr81 orient a water molecule for hydrolytic attack. The catalytic residues of the CheX-CheY3 complex are virtually superimposable on those of the Escherichia coli CheZ phosphatase complexed with CheY, even though the active site helices of CheX and CheZ are oriented nearly perpendicular to one other. Thus, evolution has found two structural solutions to achieve the same catalytic mechanism through different helical spacing and side chain lengths of the conserved acid/amide residues in CheX and CheZ.

  9. Local entropy difference upon a substrate binding of a psychrophilic α-amylase and a mesophilic homologue

    NASA Astrophysics Data System (ADS)

    Kosugi, Takahiro; Hayashi, Shigehiko

    2011-01-01

    Psychrophilic α-amylase from the antarctic bacterium pseudoalteromonashaloplanktis (AHA) and its mesophilic homologue, porcine pancreatic α-amylase (PPA) are theoretically investigated with molecular dynamics (MD) simulations. We carried out 240-ns MD simulations for four systems, AHA and PPA with/without the bound substrate, and examined protein conformational entropy changes upon the substrate binding. We developed an analysis that decomposes the entropy changes into contributions of individual amino acids, and successfully identified protein regions responsible for the entropy changes. The results provide a molecular insight into the structural flexibilities of those enzymes related to the temperature dependences of the enzymatic activity.

  10. EVIDENCE FOR BIDENTATE SUBSTRATE BINDING AS THE BASIS FOR THE K48 LINKAGE SPECIFICITY OF OTUBAIN 1

    PubMed Central

    Wang, Tao; Yin, Luming; Cooper, Eric M.; Lai, Ming-Yih; Dickey, Seth; Pickart, Cecile M.; Fushman, David; Wilkinson, Keith D.; Cohen, Robert E.; Wolberger, Cynthia

    2009-01-01

    Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination. PMID:19211026

  11. Synthesis, spectral investigations, antimicrobial activity and DNA-binding studies of novel charge transfer complex of 1,10-phenanthroline as an electron donor with π-acceptor p-Nitrophenol

    NASA Astrophysics Data System (ADS)

    Khan, Ishaat M.; Ahmad, Afaq

    2010-08-01

    Proton or charge transfer (CT) complex of donor, 1,10-phenanthroline (Phen) with π-acceptor, p-Nitrophenol (PNP) has been studied spectrophotometrically in methanol at room temperature. The binding of the CT complex with calf thymus (ct) DNA has been investigated by fluorescence spectrum, to establish the ability of the CT complex of its interaction with DNA. Stern-Volmer quenching constant ( Ksv) has also been calculated. The formation constant ( KCT), molar extinction coefficient ( ɛCT), free energy (Δ Go) and stoichiometric ratio of the CT complex have been determined by Benesi-Hildebrand equation. The stoichiometry was found to be 1:1. The CT complex was screened for its pharmacology as antibacterial and antifungal activity against various bacterial and fungal strains, showing excellent antibacterial and antifungal activity. The newly synthesized CT complex has been characterized by FTIR spectra, elemental analysis, 1H NMR, electronic absorption spectra. TGA-DTA studies were also carried out to check the stability of CT complex.

  12. Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

    PubMed Central

    Ben-Shimon, Avraham; Niv, Masha Y.

    2011-01-01

    Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions. PMID:22125489

  13. Chemopreventive Agents from Physalis minima Function as Michael Reaction Acceptors

    PubMed Central

    Men, Ruizhi; Li, Ning; Ding, Chihong; Tang, Yingzhan; Xing, Yachao; Ding, Wanjing; Ma, Zhongjun

    2016-01-01

    Background: The fruits of some varieties of genus Physalis have been used as delicious fruits and functional food in the Northeast of China. Materials and Methods: To reveal the functional material basis, we performed bioactivity-guided phytochemical research and chemopreventive effect assay of the constituents from Physalis minima. Results: It was demonstrated that the ethyl acetate extract of P. minima L. (EEPM) had potential quinone reductase (QR) inducing activity with induction ratio (IR, QR induction activity) value of 1.47 ± 0.24, and glutathione binding property as potential Michael reaction acceptors (with an α, β-unsaturated ketone moiety). Furthermore, bioactivity-guided phytochemical research led eight compounds (1–8), which were elucidated as 3-isopropyl-5-acetoxycyclohexene-2-one-1 (1), isophysalin B (2), physalin G (3), physalin D (4), physalin I (5), physordinose B (6), stigmasterol-3-O-β-D-glucopyranoside (7) and 5α-6β-dihydroxyphysalin R (8) on the basis of nuclear magnetic resonance spectroscopy analyses and HRESIMS. Then, isophysalin B (2) and physordinose B (6) showed significant QR inducing activity with IR value of 2.80 ± 0.19 and 2.38 ± 0.46, respectively. SUMMARY An ultra-performance liquid chromatographic method with glutathione as the substrate was used to detect the Michael reaction acceptors in extracts of Physalis minima (EPM)We investigated the chemical constituents of EPM guided by biological activity methodIsophysalin B (1) and physordinose B (6) showed strong quinone reductase inducing activity with induction ratio values of 2.80 ± 0.19 and 2.38 ± 0.46This study generated useful information for consumers and many encourage researchers to utilize edible fruits from Physalis as a source of phytochemicals Abbreviations used: EPM: Extracts of Physalis minima, EEPM: Ethyl acetate extract of Physalis minima L., GSH: Glutathione, MRAs: Michael reaction acceptors, QR: Quinone reductase. PMID:27279713

  14. Crystal Structure of 12-Lipoxygenase Catalytic-Domain-Inhibitor Complex Identifies a Substrate-Binding Channel for Catalysis

    SciTech Connect

    Xu, Shu; Mueser, Timothy C.; Marnett, Lawrence J.; Funk, Jr., Max O.

    2014-10-02

    Lipoxygenases are critical enzymes in the biosynthesis of families of bioactive lipids including compounds with important roles in the initiation and resolution of inflammation and in associated diseases such as diabetes, cardiovascular disease, and cancer. Crystals diffracting to high resolution (1.9 {angstrom}) were obtained for a complex between the catalytic domain of leukocyte 12-lipoxygenase and the isoform-specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP). In the three-dimensional structure of the complex, the inhibitor occupied a new U-shaped channel open at one end to the surface of the protein and extending past the redox-active iron site that is essential for catalysis. In models, the channel accommodated arachidonic acid, defining the binding site for the substrate of the catalyzed reaction. There was a void adjacent to the OPP binding site connecting to the surface of the enzyme and providing a plausible access channel for the other substrate, oxygen.

  15. Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels.

    PubMed

    McDevitt, Christopher A; Buchanan, Grant; Sargent, Frank; Palmer, Tracy; Berks, Ben C

    2006-12-01

    The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of approximately 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins. PMID:17212781

  16. The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin.

    PubMed

    Gamble, Michael; Künze, Georg; Brancale, Andrea; Wilson, Keith S; Jones, D Dafydd

    2012-01-01

    The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca(2+), are proposed to be important for ISP activity through structural changes. The presence of >0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca(2+) at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions. PMID:23650602

  17. Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates.

    PubMed

    Begley, Thomas J; Haas, Brian J; Morales, Juan C; Kool, Eric T; Cunningham, Richard P

    2003-01-01

    We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product. PMID:12509271

  18. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

    PubMed Central

    Mouchlis, Varnavas D.; Bucher, Denis; McCammon, J. Andrew; Dennis, Edward A.

    2015-01-01

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface. PMID:25624474

  19. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    PubMed

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  20. Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories.

    PubMed

    Boucher, Nathalie; Noll, Kenneth M

    2016-09-01

    The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, β-1,4-mannotriose, and β-1,4-mannotetraose. The CelE orthologs additionally bind β-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind β-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts. PMID:27457081

  1. Dynamics and structural changes induced by ATP and/or substrate binding in the inward-facing conformation state of P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Watanabe, Yurika; Hsu, Wei-Lin; Chiba, Shuntaro; Hayashi, Tomohiko; Furuta, Tadaomi; Sakurai, Minoru

    2013-02-01

    P-glycoprotein (P-gp) is a multidrug transporter that catalyzes the transport of a substrate. To elucidate the underlying mechanism of this type of substrate transport, we performed molecular dynamics (MD) simulations using the X-ray crystal structure of P-gp, which has an inward-facing conformation. Our simulations indicated that the dimerization of the nucleotide binding domains (NBDs) is driven by the binding of ATP to the NBDs and/or the binding of the substrate to a cavity in the transmembrane domains (TMDs). Based on these results, we discuss a role of ATP in the allosteric communication that occurs between the NBDs and the TMDs.

  2. Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates*S⃞

    PubMed Central

    Philominathan, Sagaya Theresa Leena; Koide, Takaki; Hamada, Kentaro; Yasui, Hiroyuki; Seifert, Soenke; Matsushita, Osamu; Sakon, Joshua

    2009-01-01

    Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca2+. We have shown that CBD can be functional to anchor growth factors in local tissue. A 1H-15N HSQC NMR titration study with three different tropocollagen analogues ((POG)10)3, ((GPOG)7PRG)3, and (GPRG(POG)7C-carbamidomethyl)3, mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)7PRG)3, (PROXYL-G(POG)7)3, and (GPRG(POG)7C-PROXYL)3 (where PROXYL represents 2,2,5,5-tetramethyl-l-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-ray scattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix. PMID:19208618

  3. A Potential Substrate Binding Conformation of β-Lactams and Insight into the Broad Spectrum of NDM-1 Activity

    PubMed Central

    Yuan, Qinghui; He, Lin

    2012-01-01

    New Delhi metallo-β-lactamase 1 (NDM-1) is a key enzyme that the pathogen Klebsiella pneumonia uses to hydrolyze almost all β-lactam antibiotics. It is currently unclear why NDM-1 has a broad spectrum of activity. Docking of the representatives of the β-lactam families into the active site of NDM-1 is reported here. All the β-lactams naturally fit the NDM-1 pocket, implying that NDM-1 can accommodate the substrates without dramatic conformation changes. The docking reveals two major binding modes of the β-lactams, which we tentatively name the S (substrate) and I (inhibitor) conformers. In the S conformers of all the β-lactams, the amide oxygen and the carboxylic group conservatively interact with two zinc ions, while the substitutions on the fused rings show dramatic differences in their conformations and positions. Since the bridging hydroxide ion/water in the S conformer is at the position for the nucleophilic attack, the S conformation may simulate the true binding of a substrate to NDM-1. The I conformer either blocks or displaces the bridging hydroxide ion/water, such as in the case of aztreonam, and is thus inhibitory. The docking also suggests that substitutions on the β-lactam ring are required for β-lactams to bind in the S conformation, and therefore, small β-lactams such as clavulanic acid would be inhibitors of NDM-1. Finally, our docking shows that moxalactam uses its tyrosyl-carboxylic group to compete with the S conformer and would thus be a poor substrate of NDM-1. PMID:22825119

  4. Tight-binding model study of substrate induced pseudo-spin polarization and magnetism in mono-layer graphene

    NASA Astrophysics Data System (ADS)

    Sahu, Sivabrata; Rout, G. C.

    2016-06-01

    We present here a tight-binding model study of generation of magnetism and pseudo-spin polarization in monolayer graphene arising due to substrate, impurity and Coulomb correlation effects. The model Hamiltonian contains the first-, second- and third-nearest-neighbor hopping integrals for π electrons of graphene besides substrate induced gap, impurity interactions and Coulomb correlation of electrons. The Hubbard type Coulomb interactions present in both the sub-lattices A and B are treated within the mean-field approximation. The electronic Green's functions are calculated by using Zubarev's technique and hence the electron occupancies of both sub-lattices are calculated for up and down spins separately. These four temperature dependent occupancies are calculated numerically and self-consistently. Then we have calculated the temperature dependent pseudo-spin polarization, ferromagnetic and anti-ferromagnetic magnetizations. We observe that there exists pseudo-spin polarization for lower Coulomb energy, u < 2.2t1 and pseudo-spin polarization is enhanced with substrate induced gap and impurity effect. For larger Coulomb energy u > 2.5t1, there exists pseudo-spin polarization (p); while ferromagnetic (m) and antiferromagnetic (pm) magnetizations exhibit oscillatory behavior. With increase of the substrate induced gap, the ferromagnetic and antiferromagnetic transition temperatures are enhanced with increase of the substrate induced gap; while polarization (p) is enhanced in magnitude only.

  5. Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

    PubMed Central

    Luo, Huei-Ru; Wu, Szu-Pei; Hsu, Chun-Hua

    2013-01-01

    Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60°C and specific activity of about 2,600 U/mg with optimal temperature of 90°C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme's activity. PMID:24386145

  6. Unveiling substrate RNA binding to H/ACA RNPs: one side fits all

    PubMed Central

    Li, Hong

    2008-01-01

    The H/ACA RNP pseudouridylases function on a large number of extraordinarily complex RNA substrates including pre-ribosomal and small nuclear RNAs. Recent structural data show that H/ACA RNPs capture their RNA substrates via a simple one-sided attachment model. However, the precise placement of each RNA substrate into the active site of the catalytic subunit relies on the essential functions of the RNP proteins. The specific roles of each HACA RNP protein are being elucidated by a combination of structural and biochemical studies. PMID:18178425

  7. High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner.

    PubMed

    Gagnon, Susannah M L; Meloncelli, Peter J; Zheng, Ruixiang B; Haji-Ghassemi, Omid; Johal, Asha R; Borisova, Svetlana N; Lowary, Todd L; Evans, Stephen V

    2015-11-01

    Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. PMID:26374898

  8. Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1.

    PubMed

    Tanui, Rose; Tao, Zhen; Silverstein, Nechama; Kanner, Baruch; Grewer, Christof

    2016-05-27

    Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse. PMID:27044739

  9. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.

    PubMed

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

    2015-06-01

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

  10. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

    PubMed Central

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

    2015-01-01

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

  11. Binding of C5-dicarboxylic substrate to aspartate aminotransferase: implications for the conformational change at the transaldimination step.

    PubMed

    Islam, Mohammad Mainul; Goto, Masaru; Miyahara, Ikuko; Ikushiro, Hiroko; Hirotsu, Ken; Hayashi, Hideyuki

    2005-06-14

    The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, l-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate, l-glutamate. In this study, we analyzed the pH-dependent binding of 2-methyl-l-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-l-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-l-aspartate and the aldimine. The structures of the Michaelis complexes of the enzyme with l-aspartate and l-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that l-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine, while that of l-aspartate is close to the aldimine. These models explain the observations for 2-methyl-l-glutamate and 2-methyl-l-aspartate. The crystal structures of the complexes of aspartate aminotransferase with phosphopyridoxyl derivatives of l-glutamate, d-glutamate, and 2-methyl-l-glutamate were solved as the models for the external aldimine and ketimine complexes of l-glutamate. All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-l-aspartate. Taking these facts altogether, it was strongly suggested that the binding of l-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are

  12. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone. PMID:23828603

  13. Substrate Pathways in the Nitrogenase MoFe Protein by Experimental Identification of Small Molecule Binding Sites

    PubMed Central

    2016-01-01

    In the nitrogenase molybdenum-iron (MoFe) protein, we have identified five potential substrate access pathways from the protein surface to the FeMo-cofactor (the active site) or the P-cluster using experimental structures of Xe pressurized into MoFe protein crystals from Azotobacter vinelandii and Clostridium pasteurianum. Additionally, all published structures of the MoFe protein, including those from Klebsiella pneumoniae, were analyzed for the presence of nonwater, small molecules bound to the protein interior. Each pathway is based on identification of plausible routes from buried small molecule binding sites to both the protein surface and a metallocluster. Of these five pathways, two have been previously suggested as substrate access pathways. While the small molecule binding sites are not conserved among the three species of MoFe protein, residues lining the pathways are generally conserved, indicating that the proposed pathways may be accessible in all three species. These observations imply that there is unlikely a unique pathway utilized for substrate access from the protein surface to the active site; however, there may be preferred pathways such as those described here. PMID:25710326

  14. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

    PubMed

    Chen, Qi; Luan, Zheng-Jiao; Yu, Hui-Lei; Cheng, Xiaolin; Xu, Jian-He

    2015-11-01

    A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications. PMID:26556053

  15. The effector domain of myristoylated alanine-rich C kinase substrate binds strongly to phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Wang, J; Arbuzova, A; Hangyás-Mihályné, G; McLaughlin, S

    2001-02-16

    Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151-175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187-26193). We report here that adding 10-100 nm MARCKS-(151-175) to a subphase containing either PLC-delta or -beta inhibits hydrolysis of PIP(2) in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP(2). Two direct binding measurements, based on centrifugation and fluorescence, show that approximately 10 nm PIP(2), in the form of vesicles containing 0.01%, 0.1%, or 1% PIP(2), binds 50% of MARCKS-(151-175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151-175) forms an electroneutral complex with approximately 4 PIP(2). MARCKS-(151-175) binds equally well to PI(4,5)P(2) and PI(3,4)P(2). Local electrostatic interactions of PIP(2) with MARCKS-(151-175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mm decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP(2) in the plasma membrane, and release the bound PIP(2) upon interaction with Ca(2+)/calmodulin or phosphorylation by protein kinase C. PMID:11053422

  16. Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis

    PubMed Central

    Otsuka, Taketo; Kirkham, Charmaine; Johnson, Antoinette; Jones, Megan M.

    2014-01-01

    Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-μg and 50-μg doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis. PMID:24914218

  17. Molecular dynamics of Mycobacterium tuberculosis KasA: implications for inhibitor and substrate binding and consequences for drug design.

    PubMed

    Schaefer, Benjamin; Kisker, Caroline; Sotriffer, Christoph A

    2011-11-01

    Inhibition of the production of fatty acids as essential components of the mycobacterial cell wall has been an established way of fighting tuberculosis for decades. However, increasing resistances and an outdated medical treatment call for the validation of new targets involved in this crucial pathway. In this regard, the β-ketoacyl ACP synthase KasA is a promising enzyme. In this study, three molecular dynamics simulations based on the wildtype crystal structures of inhibitor bound and unbound KasA were performed in order to investigate the flexibility and conformational space of this target. We present an exhaustive analysis of the binding-site flexibility and representative pocket conformations that may serve as new starting points for structure-based drug design. We also revealed a mechanism which may account for the comparatively low binding affinity of thiolactomycin. Furthermore, we examined the behavior of water molecules within the binding pocket and provide recommendations how to handle them in the drug design process. Finally, we analyzed the dynamics of a channel that accommodates the long-chain fatty acid substrates and, thereby, propose a mechanism of substrate access to this channel and how products are most likely released. PMID:22076471

  18. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    PubMed Central

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  19. Design of molecularly imprinted conducting polymer protein-sensing films via substrate-dopant binding.

    PubMed

    Komarova, Elena; Aldissi, Matt; Bogomolova, Anastasia

    2015-02-21

    Addressing the challenge of protein biosensing using molecularly imprinted polymers (MIP), we have developed and tested a novel approach to creating sensing conducive polymer films imprinted with a protein substrate, ricin toxin chain A (RTA). Our approach for creating MIP protein sensing films is based on a concept of substrate-guided dopant immobilization with subsequent conducting polymer film formation. In this proof-of-concept work we have tested three macromolecular dopants with strong protein affinity, Ponceau S, Coomassie BB R250 and ι-Carrageenan. The films were formed using sequential interactions of the substrate, dopant and pyrrole, followed by electrochemical polymerization. The films were formed on gold array electrodes allowing for extensive data acquisition. The thickness of the films was optimized to allow for efficient substrate extraction, which was removed by a combination of protease and detergent treatment. The MIP films were tested for substrate rebinding using electrochemical impedance spectroscopy (EIS). The presence of macromolecular dopants was essential for MIP film specificity. Out of three dopants tested, RTA-imprinted polypyrrole films doped with Coomassie BB performed with highest specificity towards detection of RTA with a level of detection (LOD) of 0.1 ng ml(-1). PMID:25574520

  20. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators

    PubMed Central

    Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J.; Pines, Jonathon

    2016-01-01

    The APC/C is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the Spindle Assembly Checkpoint (SAC). How the APC/C recognises its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in Cyclin A, BUBR1, BUB1 and Acm1, and show that it binds to the APC/C co-activator CDC20. The ABBA motif in Cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  1. The Crystal Structure of Escherichia coli Spermidine Synthase SpeE Reveals a Unique Substrate-binding Pocket

    SciTech Connect

    Zhou, X.; Chua, T; Tkaczuk, K; Bujnicki, J; Sivaraman, J

    2010-01-01

    Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterized, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains - a small N-terminal {beta}-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity.

  2. Characterization of a variety of standard collagen substrates: ultrastructure, uniformity, and capacity to bind and promote growth of neurons

    SciTech Connect

    Iversen, P.L.; Partlow, L.M.; Stensaas, L.J.; Moatamed, F.

    1981-06-01

    Collagen substrates were characterized after preparation by the four methods most commonly used for tissue culture (saline precipitation, exposure to ammonium hydroxide vapor, exposure to ultraviolet light, and air drying). Although roughly equivalent percentages of collagen were precipitated by each technique (87 to 97%), marked differences were found in surface uniformity and ultrastructure. Substrates were quite uniform if precipitated by exposure to ammonium hydroxide or ultraviolet light, of intermediate uniformity if saline precipitated, and not at all uniform if air dried. Scanning electron microscopy revealed that (a) ammonium hydroxide and saline precipitation primarily resulted in formation of collagen fibrils, (b) air drying produced a small number of fibrils plus a large amount of amorphous material, and (c) exposure to ultraviolet light only resulted in the formation of globular, nonfibrillar collagen aggregates. The capacity of collagen substrates to bind and grow neurons differed markedly with the method of preparation and the amount of collagen plated per unit area. Quantifications of binding and growth of both cerebral and sympathetic neurons revealed that these are separate measures of the biocompatibility of a surface and that growth was uniformly inferior on globular collagen that had been precipitated by ultraviolet light. Long-term (greater than or equal to 2 wk) growth of sympathetic neurons was optimal on thick beds of saline-precipitated collagen, whereas short-term growth was best on thin layers of either saline or ammonium hydroxide-precipitated collagen. Cerebral neurons bound and grew optimally on thick collagen beds after both short- and long-term culture. In addition, cerebral neurons were found to be more dependent on the method of precipitation of the thin collagen substrates than were sympathetic neurons.

  3. HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants.

    PubMed

    Altman, Michael D; Ali, Akbar; Reddy, G S Kiran Kumar; Nalam, Madhavi N L; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X; Gilson, Michael K; Schiffer, Celia A; Rana, Tariq M; Tidor, Bruce

    2008-05-14

    The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance. PMID:18412349

  4. HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibit Subnanomolar Binding to Drug-Resistant Variants

    PubMed Central

    Altman, Michael D.; Ali, Akbar; Reddy, G. S. Kiran Kumar; Nalam, Madhavi N. L.; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X.; Gilson, Michael K.; Schiffer, Celia A.; Rana, Tariq M.; Tidor, Bruce

    2010-01-01

    The acquisition of drug-resistance mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from Ki of 30–50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6–13 fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors — robust binders (maximum affinity loss of 14–16 fold), moderate binders (35–80 fold), and susceptible binders (greater than 100 fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance. PMID:18412349

  5. Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase.

    PubMed

    Kramhøft, Birte; Bak-Jensen, Kristian Sass; Mori, Haruhide; Juge, Nathalie; Nøhr, Jane; Svensson, Birte

    2005-02-15

    Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites. PMID:15697208

  6. Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

    SciTech Connect

    Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

    2003-08-30

    The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

  7. Identification of target messenger RNA substrates for the murine deleted in azoospermia-like RNA-binding protein.

    PubMed

    Jiao, Xinfu; Trifillis, Panayiota; Kiledjian, Megerditch

    2002-02-01

    The murine autosomal deleted in azoospermia-like protein (mDAZL) is a germ cell-restricted RNA-binding protein essential for sperm production. Homozygous disruption of the mDAZL gene results in the absence of germ cells beyond the spermatogonial stage. Progress into the function of DAZL in spermatogenesis has been hampered without identification of the cognate mRNA substrates that it binds to and regulates. Using the isolation of specific nucleic acids associated with proteins (SNAAP) technique recently developed in our lab, we identified mRNAs from testis that were specifically bound by mDAZL. One mRNA encoded the Tpx-1 protein, a testicular cell adhesion protein essential for the progression of spermatogenesis. A 26-nucleotide region necessary and sufficient to bind mDAZL was found within additional mRNAs isolated by the screen. These included mRNA encoding Pam, a protein associated with myc; GRSF1, an mRNA-binding protein involved in translation activation, and TRF2, a TATA box-binding protein-like protein involved in transcriptional regulation. Each mRNA containing the mDAZL binding site was specifically bound by mDAZL. A similar sequence is also present in the Cdc25A mRNA, a threonine/tyrosine phosphatase involved in cell cycle progression. The mDAZL and Cdc25A homologues are functionally linked in Drosophila and are necessary for spermatogenesis. Our demonstration that Tpx-1 and Cdc25A mRNAs are bound by mDAZL suggests that mDAZL regulates a subset of mRNAs necessary for germ cell development and cell cycle progression. Understanding how mDAZL regulates the target mRNAs will provide new insights into spermatogenesis, strategies for therapeutic intervention in azoospermic patients, and novel approaches for male contraception. PMID:11804965

  8. Potent inhibition of mandelate racemase by a fluorinated substrate-product analogue with a novel binding mode.

    PubMed

    Nagar, Mitesh; Lietzan, Adam D; St Maurice, Martin; Bearne, Stephen L

    2014-02-25

    Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR-TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg(2+) with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid-base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid-base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR-tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg(2+) and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our

  9. The proline-rich N-terminal sequence of calcineurin Abeta determines substrate binding.

    PubMed

    Kilka, Susann; Erdmann, Frank; Migdoll, Alexander; Fischer, Gunter; Weiwad, Matthias

    2009-03-10

    Three different genes of catalytic subunit A of the Ca(2+)-dependent serine/threonine protein phosphatase calcineurin (CaN) are encoded in the human genome forming heterodimers with regulatory subunit B. Even though physiological roles of CaN have been investigated extensively, less is known about the specific functions of the different catalytic isoforms. In this study, all human CaN holoenzymes containing either the alpha, beta, or gamma isoform of the catalytic subunit (CaN alpha, beta, or gamma, respectively) were expressed for the first time. Comparative kinetic analysis of the dephosphorylation of five specific CaN substrates provided evidence that the distinct isoforms of the catalytic subunit confer substrate specificities to the holoenzymes. CaN alpha dephosphorylates the transcription factor Elk-1 with 7- and 2-fold higher catalytic efficiencies than the beta and gamma isoforms, respectively. CaN gamma exhibits the highest k(cat)/K(m) value for DARPP-32, whereas the catalytic efficiencies for the dephosphorylation of NFAT and RII peptide were 3- and 5-fold lower, respectively, when compared with the other isoforms. Elk-1 and NFAT reporter gene activity measurements revealed even more pronounced substrate preferences of CaNA isoforms. Moreover, kinetic analysis demonstrated that CaN beta exhibits for all tested protein substrates the lowest K(m) values. Enzymatic characterization of the CaN beta(P14G/P18G) variant as well as the N-terminal truncated form CaN beta(22-524) revealed that the proline-rich sequence of CaN beta is involved in substrate recognition. CaN beta(22-524) exhibits an at least 4-fold decreased substrate affinity and a 5-fold increased turnover number. Since this study demonstrates that all CaN isoforms display the same cytoplasmic subcellular distribution and are expressed in each tested cell line, differences in substrate specificities may determine specific physiological functions of the distinct isoforms. PMID:19154138

  10. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.

    PubMed

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-07-22

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

  11. Dynamics of iron-acceptor-pair formation in co-doped silicon

    SciTech Connect

    Bartel, T.; Gibaja, F.; Graf, O.; Gross, D.; Kaes, M.; Heuer, M.; Kirscht, F.; Möller, C.; Lauer, K.

    2013-11-11

    The pairing dynamics of interstitial iron and dopants in silicon co-doped with phosphorous and several acceptor types are presented. The classical picture of iron-acceptor pairing dynamics is expanded to include the thermalization of iron between different dopants. The thermalization is quantitatively described using Boltzmann statistics and different iron-acceptor binding energies. The proper understanding of the pairing dynamics of iron in co-doped silicon will provide additional information on the electronic properties of iron-acceptor pairs and may become an analytical method to quantify and differentiate acceptors in co-doped silicon.

  12. Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Chowdhury, Rasheduzzaman; Ivison, David; Domene, Carmen; Schofield, Christopher J

    2011-09-21

    Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes. PMID:21796301

  13. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport

    SciTech Connect

    Kim, E.J.; Zhen, R.G.; Rea, P.A. )

    1994-06-21

    The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H[sup +]-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H[sup +]-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H[sup +]-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H[sup +] translocation is not known. Here the authors address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H[sup +]-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H[sup +] translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H[sub +] translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.

  14. Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.

    PubMed

    Zhu, Haixia; Xu, Guochao; Zhang, Kai; Kong, Xudong; Han, Ruizhi; Zhou, Jiahai; Ni, Ye

    2016-01-01

    Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine. PMID:27292129

  15. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.

    PubMed Central

    Kim, E J; Zhen, R G; Rea, P A

    1994-01-01

    The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H(+)-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H(+)-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H+ translocation is not known. Here we address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H+ translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H+ translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP. Images PMID:8016125

  16. Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding

    PubMed Central

    Zhu, Haixia; Xu, Guochao; Zhang, Kai; Kong, Xudong; Han, Ruizhi; Zhou, Jiahai; Ni, Ye

    2016-01-01

    Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine. PMID:27292129

  17. A study of substrate specificity for a CTD phosphatase, SCP1, by proteomic screening of binding partners.

    PubMed

    Kim, Young Jun; Bahk, Young Yil

    2014-05-30

    RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are a newly emerging family of phosphatases. Recently a CTD-specific phosphatase, small CTD phosphatase 1 (SCP1), has shown to act as an evolutionarily conserved transcriptional corepressor for inhibiting neuronal gene transcription in non-neuronal cells. In this study, using the established NIH/3T3 and HEK293T cells, which are expressing human SCP1 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for SCP1. Although the present findings provide the possibility for new avenues to provide to a better understanding of cellular physiology of SCP1, now these proteomic and some immunological approaches for SCP1 interactome might not represent the accurate physiological relevance in vivo. In this presentation, we focus the substrate specificity to delineate an appearance of the dephosphorylation reaction catalyzed by SCP1 phosphatase. We compared the phosphorylated sequences of the immunologically confirmed binding partners with SCP1 searched in HPRD. We found the similar sequences from CdcA3 and validated the efficiency of enzymatic catalysis for synthetic phosphopeptides the recombinant SCP1. This approach led to the identification of several interacting partners with SCP1. We suggest that CdcA3 could be an enzymatic substrate for SCP1 and that SCP1 might have the relationship with cell cycle regulation through enzymatic activity against CdcA3. PMID:24769477

  18. Molecular modeling and docking of novel laccase from multiple serotype of Yersinia enterocolitica suggests differential and multiple substrate binding.

    PubMed

    Singh, Deepti; Sharma, Krishna Kant; Dhar, Mahesh Shanker; Virdi, Jugsharan Singh

    2014-06-20

    Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for metal homeostasis, acquisition and oxidation. Using specific primers, yacK coding for MCO was amplified from different serotypes of Yersinia enterocolitica biovar 1A. Homology modeling of the protein followed by docking with five well-known substrates for different MCO's (viz., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid [ABTS], syringaldazine, L-tyrosine, ammonium ferrous sulfate and guaiacol), lignin monomers (Coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol) and two inhibitors i.e., kojic acid and N-hydroxyglycine was done. The docking gave maximum GoldScore i.e., 91.93 and 72.64 with ammonium ferrous sulfate and ABTS, respectively. Similarly, docking with ICM gave -82.10 and -83.61 docking score, confirming the protein to be true laccase with ferroxidase activity. Further, validation with ammonium ferrous sulfate as substrate gave laccase activity of 0.36Units/L/min. Guaiacol, L-tyrosine, and lignin monomers showed good binding affinity with protein models with GoldScores of 35.89, 41.82, 40.41, 41.12 and 43.10, respectively. The sequence study of all the cloned Yack genes showed serotype specific clade in dendrogram. There was distinct discrimination in the ligand binding affinity of Y. enterocolitica laccase, among strains of same clonal groups, suggesting it as a tool for phylogenetic studies. PMID:24832734

  19. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions. PMID:26034298

  20. A novel cyclophilin from parasitic and free-living nematodes with a unique substrate- and drug-binding domain.

    PubMed

    Ma, Dong; Nelson, Laura S; LeCoz, Krystel; Poole, Catherine; Carlow, Clotilde K S

    2002-04-26

    A highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus (OvCYP-16). This 25-kDa cyclophilin shares 43-46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans (CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu(124) (C. elegans) or Asp(123) (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A. CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism. PMID:11847225

  1. [Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase].

    PubMed

    Bakuleva, N P; Kostenko, E B; Baĭkov, A A; Avaeva, S M

    1981-05-01

    Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi. PMID:6117332

  2. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity

    PubMed Central

    Maddock, Danielle J.; Patrick, Wayne M.; Gerth, Monica L.

    2015-01-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2′-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary–secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2′-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme–cofactor interactions. PMID:26034298

  3. Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding.

    PubMed

    Svensson, B; Clarke, A J; Svendsen, I; Møller, H

    1990-02-22

    Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed. PMID:2108020

  4. An allosteric model for control of pore opening by substrate binding in the EutL microcompartment shell protein

    PubMed Central

    Thompson, Michael C; Cascio, Duilio; Leibly, David J; Yeates, Todd O

    2015-01-01

    The ethanolamine utilization (Eut) microcompartment is a protein-based metabolic organelle that is strongly associated with pathogenesis in bacteria that inhabit the human gut. The exterior shell of this elaborate protein complex is composed from a few thousand copies of BMC-domain shell proteins, which form a semi-permeable diffusion barrier that provides the interior enzymes with substrates and cofactors while simultaneously retaining metabolic intermediates. The ability of this protein shell to regulate passage of substrate and cofactor molecules is critical for microcompartment function, but the details of how this diffusion barrier can allow the passage of large cofactors while still retaining small intermediates remain unclear. Previous work has revealed two conformations of the EutL shell protein, providing substantial evidence for a gated pore that might allow the passage of large cofactors. Here we report structural and biophysical evidence to show that ethanolamine, the substrate of the Eut microcompartment, acts as a negative allosteric regulator of EutL pore opening. Specifically, a series of X-ray crystal structures of EutL from Clostridium perfringens, along with equilibrium binding studies, reveal that ethanolamine binds to EutL at a site that exists in the closed-pore conformation and which is incompatible with opening of the large pore for cofactor transport. The allosteric mechanism we propose is consistent with the cofactor requirements of the Eut microcompartment, leading to a new model for EutL function. Furthermore, our results suggest the possibility of redox modulation of the allosteric mechanism, opening potentially new lines of investigation. PMID:25752492

  5. The studies on substrate, product and inhibitor binding to a wild-type and neuronopathic form of human acid-beta-glucosidase.

    PubMed

    Zubrzycki, Igor Z; Borcz, Agnieszka; Wiacek, Magdalena; Hagner, Wojciech

    2007-11-01

    Gaucher disease is a lysosomal storage disorder caused by deficiency of human acid beta-glucosidase. Recent x-ray structural elucidation of the enzyme alone and in the presence of its inhibitor was done, which provided an excellent template for further studies on the binding of substrate, product and inhibitor. To draw correlations between the clinical manifestation of the disease driven by point mutations, L444P and L444R, and the placement and function of putative S-binding sites, the presented theoretical studies were undertaken, which comprised of molecular dynamics and molecular docking methods. The obtained results indicate the D443 and D445 residues as extremely important for physiological functionality of an enzyme. They also show, although indirectly, that binding of the substrate is influenced by an interplay of E235 and E334 residues, constituting putative substrate binding site, and the region flanked by D435 and D445 residues. PMID:17713797

  6. Crystal structures of α-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide.

    PubMed

    Zhu, Guangyu; Koszelak-Rosenblum, Mary; Malkowski, Michael G

    2013-10-01

    α-Dioxygenases (α-DOX) are heme-containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α-DOX oxygenate a variety of 14-20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro-R hydrogen from the α-carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H2 O2 ). We determined the X-ray crystal structures of wild type α-DOX from Oryza sativa, the wild type enzyme in complex with H2 O2 , and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of α-DOX such that the carboxylate forms ionic interactions with His-311 and Arg-559. Thr-316 aids in the positioning of carbon-2 for hydrogen abstraction. Twenty-five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H2 O2 structures provides insight into enzyme activation. The binding of H2 O2 at the distal face of the heme displaces residues His-157, Asp-158, and Trp-159 ≈ 2.5 Å from their positions in the wild type structure. As a result, the Oδ2 atom of Asp-158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp-159 and Trp-213 reorient, and the guanidinium group of Arg-559 is repositioned near Tyr-379, poised to interact with the carboxylate group of the substrate. PMID:23934749

  7. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

    PubMed Central

    Broussard, Tyler C.; Pakhomova, Svetlana; Neau, David B.; Bonnot, Ross; Waldrop, Grover L.

    2015-01-01

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin. PMID:26020841

  8. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

    PubMed

    Broussard, Tyler C; Pakhomova, Svetlana; Neau, David B; Bonnot, Ross; Waldrop, Grover L

    2015-06-23

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin. PMID:26020841

  9. Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding.

    PubMed

    Noguchi, Junji; Chaen, Kimiko; Vu, Nhuan Thi; Akasaka, Taiki; Shimada, Hiroaki; Nakashima, Takashi; Nishi, Aiko; Satoh, Hikaru; Omori, Toshiro; Kakuta, Yoshimitsu; Kimura, Makoto

    2011-08-01

    Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 Å by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase. PMID:21493662

  10. Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian Cytochrome P450

    PubMed Central

    Mak, Piotr J.; Denisov, Ilia G.; Grinkova, Yelena V.; Sligar, Stephen G.; Kincaid, James R.

    2011-01-01

    Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates; bromocriptine (BC), erythromycin (ERY) and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While, ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome. PMID:21207936

  11. The RNA-binding protein RNP29 is an unusual Toc159 transport substrate.

    PubMed

    Grimmer, Julia; Rödiger, Anja; Hoehenwarter, Wolfgang; Helm, Stefan; Baginsky, Sacha

    2014-01-01

    The precursors of RNP29 and Ferredoxin (Fd2) were previously identified in the cytosol of ppi2 plant cells with their N-terminal amino acid acetylated. Here, we explore whether precursor accumulation in ppi2 is characteristic for Toc159 client proteins, by characterizing the import properties of the RNP29 precursor in comparison to Fd2 and other Toc159-dependent or independent substrates. We find specific accumulation of the RNP29 precursor in ppi2 but not in wild type or ppi1 protoplasts. With the exception of Lhcb4, precursor accumulation is also detected with all other tested constructs in ppi2. However, RNP29 is clearly different from the other proteins because only precursor but almost no mature protein is detectable in protoplast extracts. Co-transformation of RNP29 with Toc159 complements its plastid import, supporting the hypothesis that RNP29 is a Toc159-dependent substrate. Exchange of the second amino acid in the RNP29 transit peptide to Glu or Asn prevents methionine excision but not N-terminal acetylation, suggesting that different N-acetyltransferases may act on chloroplast precursor proteins in vivo. All different RNP29 constructs are efficiently imported into wild type but not into ppi2 plastids, arguing for a minor impact of the N-terminal amino acid on the import process. PMID:24982663

  12. Structure and substrate ion binding in the sodium/proton antiporter PaNhaP

    PubMed Central

    Wöhlert, David; Kühlbrandt, Werner; Yildiz, Özkan

    2014-01-01

    Sodium/proton antiporters maintain intracellular pH and sodium levels. Detailed structures of antiporters with bound substrate ions are essential for understanding how they work. We have resolved the substrate ion in the dimeric, electroneutral sodium/proton antiporter PaNhaP from Pyrococcus abyssi at 3.2 Å, and have determined its structure in two different conformations at pH 8 and pH 4. The ion is coordinated by three acidic sidechains, a water molecule, a serine and a main-chain carbonyl in the unwound stretch of trans-membrane helix 5 at the deepest point of a negatively charged cytoplasmic funnel. A second narrow polar channel may facilitate proton uptake from the cytoplasm. Transport activity of PaNhaP is cooperative at pH 6 but not at pH 5. Cooperativity is due to pH-dependent allosteric coupling of protomers through two histidines at the dimer interface. Combined with comprehensive transport studies, the structures of PaNhaP offer unique new insights into the transport mechanism of sodium/proton antiporters. DOI: http://dx.doi.org/10.7554/eLife.03579.001 PMID:25426802

  13. Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Seo, Susan M; Birukou, Ivan; Brennan, Richard G; Kaatz, Glenn W

    2015-03-01

    The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates. PMID:25583977

  14. Binding of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein (MRP) to vesicular phospholipid membranes.

    PubMed Central

    Vergères, G; Ramsden, J J

    1998-01-01

    The myristoylated alanine-rich C kinase substrate (MARCKS) protein family has two known members, MARCKS itself and MARCKS-related protein (MRP, also called MacMARCKS or F52). They are essential for brain development and are believed to regulate the structure of the actin cytoskeleton at the plasma membrane. Hence membrane binding is central to their function. MARCKS has been quite extensively characterized; MRP much less so. Despite the fact that MRP is only two thirds the size of MARCKS, it has hitherto been assumed that the two proteins have similar properties. Here we make a detailed study, including the effects of myristoylation, lipid composition, calmodulin and phosphorylation of the binding of MRP to phospholipid vesicles. We show that both the N-terminal myristoyl moiety and the central effector domain mediate binding. MRP behaves like MARCKS in the presence of neutral phospholipids. In contrast to MARCKS, however, the incorporation of 20% of negatively-charged phospholipids only marginally increases the affinity of myristoylated MRP. Co-operativity between the myristoyl moiety and the effector domain of MRP is weak and the protein has a significantly lower affinity for these vesicles compared with MARCKS. Furthermore, calmodulin or phosphorylation of the effector domain by the catalytic subunit of protein kinase C do not significantly decrease the binding of myristoylated MRP to negatively-charged phospholipid vesicles. Our results show that the mechanisms regulating the interactions of MARCKS and MRP with phospholipid vesicles are, at least quantitatively, different. In agreement with cellular studies, we therefore propose that MARCKS and MRP have different subcellular localization and, consequently, different functions. PMID:9461483

  15. A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

    PubMed Central

    Tanaka, Takeshi; Fujiwara, Shinsuke; Nishikori, Shingo; Fukui, Toshiaki; Takagi, Masahiro; Imanaka, Tadayuki

    1999-01-01

    We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. PMID:10583986

  16. Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases

    SciTech Connect

    Wu,H.; Dombrovsky, L.; Tempel, W.; Martin, F.; Loppnau, P.; Goodfellow, G.; Grant, D.; Plotnikov, A.

    2007-01-01

    The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9- Angstroms resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.

  17. Unique Peptide Substrate Binding Properties of 110-kDa Heat-shock Protein (Hsp110) Determine Its Distinct Chaperone Activity*

    PubMed Central

    Xu, Xinping; Sarbeng, Evans Boateng; Vorvis, Christina; Kumar, Divya Prasanna; Zhou, Lei; Liu, Qinglian

    2012-01-01

    The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s. PMID:22157767

  18. The effect of the carbohydrate binding module on substrate degradation by the human chitotriosidase.

    PubMed

    Stockinger, Linn Wilhelmsen; Eide, Kristine Bistrup; Dybvik, Anette Israelsen; Sletta, Håvard; Vårum, Kjell Morten; Eijsink, Vincent G H; Tøndervik, Anne; Sørlie, Morten

    2015-10-01

    Human chitotriosidase (HCHT) is one of two active glycoside hydrolase family 18 chitinases produced by humans. The enzyme is associated with several diseases and is thought to play a role in the anti-parasite responses of the innate immune system. HCHT occurs in two isoforms, one 50 kDa (HCHT50) and one 39 kDa variant (HCHT39). Common for both isoforms is a catalytic domain with the (β/α)8 TIM barrel fold. HCHT50 has an additional linker-region, followed by a C-terminal carbohydrate-binding module (CBM) classified as CBM family 14 in the CAZy database. To gain further insight into enzyme functionality and especially the effect of the CBM, we expressed both isoforms and compared their catalytic properties on chitin and high molecular weight chitosans. HCHT50 degrades chitin faster than HCHT39 and much more efficiently. Interestingly, both HCHT50 and HCHT39 show biphasic kinetics on chitosan degradation where HCHT50 is faster initially and HCHT39 is faster in the second phase. Moreover, HCHT50 produces distinctly different oligomer distributions than HCHT39. This is likely due to increased transglycosylation activity for HCHT50 due the CBM extending the positive subsites binding surface and therefore promoting transglycosylation. Finally, studies with both chitin and chitosan showed that both isoforms have a similarly low degree of processivity. Combining functional and structural features of the two isoforms, it seems that HCHT combines features of exo-processive and endo-nonprocessive chitinases with the somewhat unusual CBM14 to reach a high degree of efficiency, in line with its alleged physiological task of being a "complete" chitinolytic machinery by itself. PMID:26116146

  19. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs.

    PubMed

    Alian, Akram; Griner, Sarah L; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D; Stroud, Robert M

    2009-05-19

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  20. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  1. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  2. Spectral, thermal and kinetic studies of charge-transfer complexes formed between the highly effective antibiotic drug metronidazole and two types of acceptors: σ- and π-acceptors.

    PubMed

    Refat, Moamen S; Saad, Hosam A; Adam, Abdel Majid A

    2015-04-15

    Understanding the interaction between drugs and small inorganic or organic molecules is critical in being able to interpret the drug-receptor interactions and acting mechanism of these drugs. A combined solution and solid state study was performed to describe the complexation chemistry of drug metronidazole (MZ) which has a broad-spectrum antibacterial activity with two types of acceptors. The acceptors include, σ-acceptor (i.e., iodine) and π-acceptors (i.e., dichlorodicyanobenzoquinone (DDQ), chloranil (CHL) and picric acid (PA)). The molecular structure, spectroscopic characteristics, the binding modes as well as the thermal stability were deduced from IR, UV-vis, (1)H NMR and thermal studies. The binding ratio of complexation (MZ: acceptor) was determined to be 1:2 for the iodine acceptor and 1:1 for the DDQ, CHL or PA acceptor, according to the CHN elemental analyses and spectrophotometric titrations. It has been found that the complexation with CHL and PA acceptors increases the values of enthalpy and entropy, while the complexation with DDQ and iodine acceptors decreases the values of these parameters compared with the free MZ donor. PMID:25677533

  3. Spectral, thermal and kinetic studies of charge-transfer complexes formed between the highly effective antibiotic drug metronidazole and two types of acceptors: σ- and π-acceptors

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; Saad, Hosam A.; Adam, Abdel Majid A.

    2015-04-01

    Understanding the interaction between drugs and small inorganic or organic molecules is critical in being able to interpret the drug-receptor interactions and acting mechanism of these drugs. A combined solution and solid state study was performed to describe the complexation chemistry of drug metronidazole (MZ) which has a broad-spectrum antibacterial activity with two types of acceptors. The acceptors include, σ-acceptor (i.e., iodine) and π-acceptors (i.e., dichlorodicyanobenzoquinone (DDQ), chloranil (CHL) and picric acid (PA)). The molecular structure, spectroscopic characteristics, the binding modes as well as the thermal stability were deduced from IR, UV-vis, 1H NMR and thermal studies. The binding ratio of complexation (MZ: acceptor) was determined to be 1:2 for the iodine acceptor and 1:1 for the DDQ, CHL or PA acceptor, according to the CHN elemental analyses and spectrophotometric titrations. It has been found that the complexation with CHL and PA acceptors increases the values of enthalpy and entropy, while the complexation with DDQ and iodine acceptors decreases the values of these parameters compared with the free MZ donor.

  4. Decomposition of plant materials in marine sediment exposed to different electron acceptors (O 2, NO 3-, and SO 42-), with emphasis on substrate origin, degradation kinetics, and the role of bioturbation

    NASA Astrophysics Data System (ADS)

    Kristensen, Erik; Holmer, Marianne

    2001-02-01

    Carbon mineralization of fresh and aged diatoms ( Skeletonema costatum) and barley hay ( Hordeum vulgare) was followed for 23 to 35 d in sandy and silty sediment. By the use of a thin-layer flow-through technique, it was possible to expose the sediment selectively for oxygen, nitrate or sulfate as electron acceptors in the terminal oxidation of organic carbon. Decomposition took place in two basic stages. Mineralization of the rapidly leachable fraction of the fresh materials occurred rapidly and with the same constant rate regardless of the electron acceptor available, indicating that the dissolved organic carbon released initially was labile and readily available for all heterotrophic respirers. In the case of diatoms, decay of the remaining, more refractory, particulate fraction of fresh and aged diatoms were strikingly similar, although both were degraded 5 to 10 times faster under oxic than anoxic conditions. Most of the particulate remains of diatoms after leaching apparently belong to one fraction, which maintains the same degradability even after prolonged aging. With respect to hay, the late divergence in rates of aerobic and anaerobic decay (a factor of 4 to 5 for aged hay only after 20 d) indicated that the larger hay particles (<500 μm) became exhausted in labile organic matter much slower through time than fine-particulate diatoms (˜20 μm). Anaerobic carbon mineralization rates of diatoms and hay particulates with sulfate and nitrate as electron acceptors were similar or up to two times faster with sulfate. The generally low levels of dissolved organic carbon in all incubations after the initial leaching phase suggest that the limiting step of decomposition under both aerobic and anaerobic decay is the initial hydrolytic attack on the complex particulate remains. Based on a volumetric model, we show that the exposure of anoxic subsurface sediment containing partly degraded organic material to oxygen via irrigated worm burrows or by reworking may

  5. Modulation of Heme/Substrate Binding Cleft of Neuronal Nitric-oxide Synthase (nNOS) Regulates Binding of Hsp90 and Hsp70 Proteins and nNOS Ubiquitination*

    PubMed Central

    Peng, Hwei-Ming; Morishima, Yoshihiro; Pratt, William B.; Osawa, Yoichi

    2012-01-01

    Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885–22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with NG-nitro-l-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca2+-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination. PMID:22128174

  6. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.

    PubMed

    Di Fiore, Barbara; Davey, Norman E; Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J; Pines, Jonathon

    2015-02-01

    The anaphase-promoting complex or cyclosome (APC/C) is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the spindle assembly checkpoint (SAC). How the APC/C recognizes its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in cyclin A, BUBR1, BUB1, and Acm1, and we show that it binds to the APC/C coactivator CDC20. The ABBA motif in cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  7. Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

    PubMed

    Korwar, Sudha; Morris, Benjamin L; Parikh, Hardik I; Coover, Robert A; Doughty, Tyler W; Love, Ian M; Hilbert, Brendan J; Royer, William E; Kellogg, Glen E; Grossman, Steven R; Ellis, Keith C

    2016-06-15

    C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer. PMID:27156192

  8. Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding.

    PubMed

    Fredslund, Folmer; Hachem, Maher Abou; Larsen, René Jonsgaard; Sørensen, Pernille Gerd; Coutinho, Pedro M; Lo Leggio, Leila; Svensson, Birte

    2011-09-23

    Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite -1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)(8)-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36. PMID:21827767

  9. An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

    PubMed Central

    Chiappori, Federica; Merelli, Ivan; Milanesi, Luciano; Colombo, Giorgio; Morra, Giulia

    2016-01-01

    The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery. PMID:27025773

  10. Methionine sulfoxide reductase: chemistry, substrate binding, recycling process and oxidase activity.

    PubMed

    Boschi-Muller, Sandrine; Branlant, Guy

    2014-12-01

    Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form. PMID:25108804

  11. Changes in Dynamics upon Oligomerization Regulate Substrate Binding and Allostery in Amino Acid Kinase Family Members

    PubMed Central

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2011-01-01

    Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions) favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities. PMID:21980279

  12. An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features.

    PubMed

    De Paris, Renata; Quevedo, Christian V; Ruiz, Duncan D A; Norberto de Souza, Osmar

    2015-01-01

    Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward's, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill

  13. An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features

    PubMed Central

    Ruiz, Duncan D. A.; Norberto de Souza, Osmar

    2015-01-01

    Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward’s, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill

  14. Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.

    PubMed

    Bebrone, Carine; Anne, Christine; Kerff, Frédéric; Garau, Gianpiero; De Vriendt, Kris; Lantin, Raphaël; Devreese, Bart; Van Beeumen, Jozef; Dideberg, Otto; Frère, Jean-Marie; Galleni, Moreno

    2008-08-15

    The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding. PMID:18498253

  15. Substrate Recognition by β-Ketoacyl-ACP Synthases†

    PubMed Central

    Borgaro, Janine G.; Chang, Andrew; Machutta, Carl A.; Zhang, Xujie; Tonge, Peter J.

    2011-01-01

    β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14 residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the kcat/Km for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

  16. Substrate recognition by β-ketoacyl-ACP synthases.

    PubMed

    Borgaro, Janine G; Chang, Andrew; Machutta, Carl A; Zhang, Xujie; Tonge, Peter J

    2011-12-13

    β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

  17. Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase

    SciTech Connect

    Li, Lenong; Chang, Zhenzhan; Pan, Zhiqiang; Fu, Zheng-Qing; Wang, Xiaoqiang

    2009-01-12

    Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between {beta}3-2 and {beta}3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.

  18. A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases.

    PubMed

    Giuseppe, Priscila Oliveira de; Santos, Marcelo Leite Dos; Sousa, Sylvia Morais de; Koch, Karen E; Yunes, José Andrés; Aparicio, Ricardo; Murakami, Mario Tyago

    2016-06-10

    Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs. PMID:27154221

  19. Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling).

    PubMed

    Narasimhulu, Shakunthala

    2007-03-01

    The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O(2) and specific electron transport system. The ternary complex S-Fe(+)O(2) with substrate and O(2) bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L(6) position of the iron (Site II) to which O(2) binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L(6) position of the iron. The substrate camphor binds to Site I, and releases the L(6) water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its -OH group forming Fe-O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub

  20. High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70.

    PubMed

    Morshauser, R C; Hu, W; Wang, H; Pang, Y; Flynn, G C; Zuiderweg, E R

    1999-06-25

    The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383-540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 A for the backbone atoms and 0.76 A for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions. PMID:10373374

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

    PubMed

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

    2003-08-15

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

  2. Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2: A molecular dynamics Study.

    PubMed

    Yotmanee, Pathumwadee; Rungrotmongkol, Thanyada; Wichapong, Kanin; Choi, Sy Bing; Wahab, Habibah A; Kungwan, Nawee; Hannongbua, Supot

    2015-07-01

    The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of <-10 kcal mol(-1). Besides, the P(3), P(1)', P(2)' and P(4)' subsites showed a less contribution in binding interaction (<-2 kcal mol(-1)). The catalytic water was detected nearby the carbonyl oxygen of the P(1) reacting center of the capsid, intNS3, 2A/2B and 4B/5 peptides. These results led to the order of absolute binding free energy (ΔGbind) between these substrates and the NS2B/NS3 protease ranked as capsid>intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values. PMID:26086900

  3. A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding

    PubMed Central

    2013-01-01

    Background Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42. Results Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity. Conclusions Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan. PMID:24041306

  4. Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2.

    PubMed

    Permanasari, Etin-Diah; Yasukawa, Kiyoshi; Kanaya, Shigenori

    2015-06-01

    Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg(2+) or Mn(2+) ions. The isolated RNase H domain of HIV-1 RT (RNH(HIV)) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNH(HIV) is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNH(HIV) and BstNTD-RNH(HIV), were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNH(HIV) and exhibited enzymatic activity in the presence of Mn(2+) ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg(2+) ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn(2+)-dependent activity of RNH(HIV) but do not restore the Mg(2+)-dependent activity of RNH(HIV). PMID:25673083

  5. Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions.

    PubMed Central

    Sugimoto, N; Tomka, M; Kierzek, R; Bevilacqua, P C; Turner, D H

    1989-01-01

    The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotides or water. The kinetics of circle opening as a function of substrate and Mg2+ concentrations have been measured for dCrU, rCdU, dCdT, and H2O addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg2+ binding. Evidently, the binding site for a required Mg2+ ion is dependent on both the ribozyme and the dimer substrate. The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction. Images PMID:2643083

  6. Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

    PubMed Central

    Cheng, Victor W.T.; Piragasam, Ramanaguru Siva; Rothery, Richard A.; Maklashina, Elena; Cecchini, Gary; Weiner, Joel H.

    2016-01-01

    The Complex II family of enzymes, comprising the respiratory succinate dehydrogenases and fumarate reductases, catalyze reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, the soluble fumarate reductases (e.g. that from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and the FAD was examined. The variants SdhA-R286A/K/Y and -H242A/Y, that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in assembly of a noncovalent FAD cofactor, which led to a significant decrease (−87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The “free” and “occupied” states of the active site were linked to the reduced and oxidized states of the FAD, respectively. Our data allows for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD. PMID:25569225

  7. Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates.

    PubMed

    Asbóth, B; Stokum, E; Khan, I U; Polgár, L

    1985-01-29

    To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases. PMID:3888259

  8. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

    PubMed Central

    Alian, Akram; Griner, Sarah L.; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D.; Stroud, Robert M.

    2009-01-01

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN–DNA complexes that form disulfide linkages between 5′-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  9. Redox state of flavin adenine dinucleotide drives substrate binding and product release in Escherichia coli succinate dehydrogenase.

    PubMed

    Cheng, Victor W T; Piragasam, Ramanaguru Siva; Rothery, Richard A; Maklashina, Elena; Cecchini, Gary; Weiner, Joel H

    2015-02-01

    The Complex II family of enzymes, comprising respiratory succinate dehydrogenases and fumarate reductases, catalyzes reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, soluble fumarate reductases (e.g., those from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and FAD was examined. Variants SdhA-R286A/K/Y and -H242A/Y that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in the assembly of a noncovalent FAD cofactor, which led to a significant decrease (-87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The "free" and "occupied" states of the active site were linked to the reduced and oxidized states of FAD, respectively. Our data allow for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD. PMID:25569225

  10. Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study.

    PubMed Central

    Chen, L X; Longworth, J W; Fleming, G R

    1987-01-01

    The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A. PMID:3038204

  11. Targeting bacterial membranes: NMR spectroscopy characterization of substrate recognition and binding requirements of D-arabinose-5-phosphate isomerase.

    PubMed

    Airoldi, Cristina; Sommaruga, Silvia; Merlo, Silvia; Sperandeo, Paola; Cipolla, Laura; Polissi, Alessandra; Nicotra, Francesco

    2010-02-01

    Lipopolysaccharide (LPS) is an essential component of the outer membrane of gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O-antigen. The inner-core region is highly conserved and contains at least one residue of 3-deoxy-D-manno-octulosonate (Kdo). Arabinose-5-phosphate isomerase (API) is an aldo-keto isomerase catalyzing the reversible isomerization of D-ribulose-5-phosphate (Ru5P) to D-arabinose-5-phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and gram-negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed. PMID:20039350

  12. Evaluation of Substrate and Inhibitor Binding to Yeast and Human Isoprenylcysteine Carboxyl Methyltransferases (Icmts) using Biotinylated Benzophenone-containing Photoaffinity Probes

    PubMed Central

    Hahne, Kalub; Vervacke, Jeffery; Shrestha, Liza; Donelson, James L.; Gibbs, Richard A.; Distefano, Mark D.; Hrycyna, Christine A.

    2013-01-01

    Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and S. cerevisae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition and that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. These data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites. PMID:22634004

  13. Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

    PubMed

    Flannelly, David F; Aoki, Thalia G; Aristilde, Ludmilla

    2015-09-01

    The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs. PMID:26160737

  14. Acceptors in ZnO

    SciTech Connect

    McCluskey, Matthew D. Corolewski, Caleb D.; Lv, Jinpeng; Tarun, Marianne C.; Teklemichael, Samuel T.; Walter, Eric D.; Norton, M. Grant; Harrison, Kale W.; Ha, Su

    2015-03-21

    Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence indicates these point defects have acceptor levels 3.2, 1.4, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO{sub 2} contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peak in ZnO nanocrystals is attributed to an acceptor, which may involve a Zn vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g{sub ⊥} = 2.0015 and g{sub //} = 2.0056, along with an isotropic center at g = 2.0035.

  15. Acceptors in ZnO

    SciTech Connect

    Mccluskey, Matthew D.; Corolewski, Caleb; Lv, Jinpeng; Tarun, Marianne C.; Teklemichael, Samuel T.; Walter, Eric D.; Norton, M. G.; Harrison, Kale W.; Ha, Su Y.

    2015-03-21

    Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence shows that these point defects have acceptor levels 3.2, 1.5, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO2 contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peak in ZnO nanocrystals has been attributed to an acceptor, which may involve a zinc vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g = 2.0033 and g = 2.0075, along with an isotropic center at g = 2.0053.

  16. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    SciTech Connect

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate

  17. Structural Basis for Substrate Specificity in Phosphate Binding (β/α)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12†

    PubMed Central

    Chan, Kui K.; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Gerlt, John A.

    2008-01-01

    Enzymes that share the (β/α)8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal (β/α)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of D-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates D-ribulose 5-phosphate and D-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493–2503]. We now report functional and structural studies of D-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates D-allulose 6-phosphate and D-fructose 6-phosphate in a catabolic pathway for D-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other’s substrate. The active sites (RPE complexed with D-xylitol 5-phosphate and ALSE complexed with D-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth β-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ΔT196, ΔS197 and ΔG198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that D-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group

  18. Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

    PubMed

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

    2016-09-13

    Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli. PMID:27582465

  19. Determinants of Cytochrome P450 2C8 Substrate Binding: Structures of Complexes With Montelukast, Troglitazone, Felodipine And 9-CIS-Retinoic Acid

    SciTech Connect

    Schoch, G.A.; Yano, J.K.; Dansette, P.M.; Stout, C.D.; Johnson, E.F.

    2009-05-27

    Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 {angstrom}), troglitazone (2.7 {angstrom}), felodipine (2.3 {angstrom}), and 9-cis-retinoic acid (2.6 {angstrom}) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.

  20. Acceptors in bulk and nanoscale ZnO

    NASA Astrophysics Data System (ADS)

    McCluskey, M. D.

    2012-02-01

    Zinc oxide (ZnO) is a semiconductor that emits bright UV light, with little wasted heat. This intrinsic feature makes it a promising material for energy-efficient white lighting, nano-lasers, and other optical applications. For devices to be competitive, however, it is necessary to develop reliable p-type doping. Although substitutional nitrogen has been considered as a potential p-type dopant for ZnO, theoretical and experimental work indicates that nitrogen is a deep acceptor and will not lead to p-type conductivity. This talk will highlight recent experiments on ZnO:N at low temperatures. A red/near-IR photoluminescence (PL) band is correlated with the presence of deep nitrogen acceptors. PL excitation (PLE) measurements show an absorption threshold of 2.26 eV, in good agreement with theory. Magnetic resonance experiments provide further evidence for this assignment. The results of these studies seem to rule out group-V elements as shallow acceptors in ZnO, contradicting numerous reports in the literature. If these acceptors do not work as advertised, is there a viable alternative? Optical studies on ZnO nanocrystals show some intriguing leads. At liquid-helium temperatures, a series of sharp IR absorption peaks arise from an unknown acceptor impurity. The data are consistent with a hydrogenic acceptor 0.46 eV above the valence band edge. While this binding energy is still too deep for many practical applications, it represents a significant improvement over the ˜ 1.3 eV binding energy for nitrogen acceptors. Nanocrystals present another twist. Due to their high surface-to-volume ratio, surface states are especially important. Specifically, electron-hole recombination at the surface give rises to a red luminescence band. From our PL and IR experiments, we have developed a ``unified'' model that attempts to explain acceptor and surface states in ZnO nanocrystals. This model could provide a useful framework for designing future nanoscale ZnO devices.

  1. Probing the role of aromatic residues at the secondary saccharide binding sites of human salivary α-amylase in substrate hydrolysis and bacterial binding

    PubMed Central

    Ragunath, Chandran; Manuel, Suba G.A.; Venkataraman, Venkat; Sait, Hameetha B.R.; Kasinathan, Chinnasamy; Ramasubbu, Narayanan

    2008-01-01

    SUMMARY Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: 1) hydrolysis of starch; 2) binding to hydroxyapatite; and 3) binding to bacteria (e.g. viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A and W284A), double (Y276A/W284A and W316A/W388A) or multiple (HSAmy-ar; W134A/W203A/Y276A/W284A/W316A/W388A) mutations. The crystal structure of HSAmy-ar was determined at a resolution of 1.5 Å as an acarbose complex and compared with the existing wild type acarbose complex. The wild type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch binding, hydroxyapatite and bacterial binding activities. Our results clearly showed that 1) mutation of aromatic residues does not alter the overall conformation of the molecule; 2) the single or double mutants showed either moderate or minimal changes in both starch and bacterial binding activities activity whereas the HSAmy-ar showed significant reduction in these activities; 3) the starch hydrolytic activity was reduced 10-fold in HSAmy-ar; 4) oligosaccharide hydrolytic activity was reduced in all the mutants but the action pattern was similar to that of the wild type enzyme; and 5) the hydroxyaptite binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide binding sites in HSAmy play a critical role in bacterial binding and starch

  2. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

    PubMed Central

    Padlan, Camille S.; Malashkevich, Vladimir N.; Almo, Steve C.; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E.

    2014-01-01

    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid–protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer–protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein–nucleic acid interface; (1) only 410 Å2 of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na+ stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE–lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform. PMID:24570482

  3. Molecular dynamics investigations of regioselectivity of anionic/aromatic substrates by a family of enzymes: a case study of diclofenac binding in CYP2C isoforms.

    PubMed

    Cui, Ying-Lu; Xu, Fang; Wu, Rongling

    2016-06-29

    The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided. PMID:27302079

  4. The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1

    PubMed Central

    Herbert, Alan; Rich, Alexander

    2001-01-01

    RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5′ to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11–15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited. PMID:11593027

  5. Crystal Structure of Vancosaminyltransferase GtfD from the Vancomycin Biosynthetic Pathway: Interactions with Acceptor and Nucleotide Ligands

    SciTech Connect

    Mulichak, A.M.; Lu, W.; Losey, H.C.; Walsh, C.T.; Garavito, R.M.

    2010-03-08

    The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.0 {angstrom} resolution. GtfD, a member of the bidomain GT-B glycosyltransferase superfamily, binds TDP in the interdomain cleft, while the aglycone acceptor binds in a deep crevice in the N-terminal domain. However, the two domains are more interdependent in terms of substrate binding and overall structure than was evident in the structures of closely related glycosyltransferases GtfA and GtfB. Structural and kinetic analyses support the identification of Asp13 as a catalytic general base, with a possible secondary role for Thr10. Several residues have also been identified as being involved in donor sugar binding and recognition.

  6. Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding.

    PubMed

    Peleh, Valentina; Cordat, Emmanuelle; Herrmann, Johannes M

    2016-01-01

    Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a 'holding trap' rather than a 'folding trap' mechanism. PMID:27343349

  7. Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

    PubMed Central

    Peleh, Valentina; Cordat, Emmanuelle; Herrmann, Johannes M

    2016-01-01

    Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism. DOI: http://dx.doi.org/10.7554/eLife.16177.001 PMID:27343349

  8. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    PubMed Central

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

  9. Carboxymethyl cellulose binding to mineral substrates: characterization by atomic force microscopy-based force spectroscopy and quartz-crystal microbalance with dissipation monitoring.

    PubMed

    Pensini, Erica; Yip, Christopher M; O'Carroll, Denis; Sleep, Brent E

    2013-07-15

    The attachment of the sodium salt of carboxymethyl cellulose (CMC) onto iron oxide and various silicate substrates in aqueous solution as a function of salt concentration and pH was studied by atomic force microscopy-based force spectroscopy (AFM) and quartz-crystal microbalance with dissipation monitoring (QCM-D). Both ionic strength and cation valency were found to influence substrate binding. Notably, QCM-D experiments strongly suggested that the solubility of CMC is directly impacted by the presence of CaCl2. Such data are critical for the design of new molecules for stabilizing mineral floc dispersions and for assessing the mobility of CMC-coated particles in the subsurface. Modeling of AFM data with an extended Ohshima theory showed that van der Waals and steric forces played a major role in the interactions between CMC and mineral substrates, and that hydration forces were also important. PMID:23643251

  10. Biocatalytic properties and substrate-binding ability of a modular GH10 β-1,4-xylanase from an insect-symbiotic bacterium, Streptomyces mexicanus HY-14.

    PubMed

    Kim, Do Young; Shin, Dong-Ha; Jung, Sora; Lee, Jong Suk; Cho, Han-Young; Bae, Kyung Sook; Sung, Chang-Keun; Rhee, Young Ha; Son, Kwang-Hee; Park, Ho-Yong

    2014-10-01

    The gene (1350-bp) encoding a modular β-1,4-xylanase (XylU), which consists of an N-terminal catalytic GH10 domain and a C-terminal carbohydrate-binding module 2 (CBM 2), from Streptomyces mexicanus HY-14 was cloned and functionally characterized. The purified His-tagged recombinant enzyme (rXylU, 44.0 kDa) was capable of efficiently hydrolyze diverse xylosidic compounds, p-nitrophenyl-cellobioside, and p-nitrophenyl-xylopyranoside when incubated at pH 5.5 and 65°C. Especially, the specific activities (649.8 U/mg and 587.0 U/mg, respectively) of rXylU toward oat spelts xylan and beechwood xylan were relatively higher than those (<500.0 U/mg) of many other GH10 homologs toward the same substrates. The results of enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) revealed that rXylU preferentially hydrolyzed the substrates to xylobiose (>75%) as the primary degradation product. Moreover, a small amount (4%<) of xylose was detected as the degradation product of the evaluated xylosidic substrates, indicating that rXylU was a peculiar GH10 β-1,4-xylanase with substrate specificity, which was different from its retaining homologs. A significant reduction of the binding ability of rXylU caused by deletion of the C-terminal CBM 2 to various insoluble substrates strongly suggested that the additional domain might considerably contribute to the enzyme-substrate interaction. PMID:25269606

  11. Quantum confined acceptors and donors in InSe nanosheets

    SciTech Connect

    Mudd, G. W.; Patanè, A. Makarovsky, O.; Eaves, L.; Kudrynskyi, Z. R.; Kovalyuk, Z. D.; Fay, M. W.; Zólyomi, V.; Falko, V.

    2014-12-01

    We report on the radiative recombination of photo-excited carriers bound at native donors and acceptors in exfoliated nanoflakes of nominally undoped rhombohedral γ-polytype InSe. The binding energies of these states are found to increase with the decrease in flake thickness, L. We model their dependence on L using a two-dimensional hydrogenic model for impurities and show that they are strongly sensitive to the position of the impurities within the nanolayer.

  12. Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

    SciTech Connect

    Kim, M.H.; Neubig, R.R.

    1986-03-05

    High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonist (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.

  13. Quantum computing with acceptor spins in silicon

    NASA Astrophysics Data System (ADS)

    Salfi, Joe; Tong, Mengyang; Rogge, Sven; Culcer, Dimitrie

    2016-06-01

    The states of a boron acceptor near a Si/SiO2 interface, which bind two low-energy Kramers pairs, have exceptional properties for encoding quantum information and, with the aid of strain, both heavy hole and light hole-based spin qubits can be designed. Whereas a light-hole spin qubit was introduced recently (arXiv:1508.04259), here we present analytical and numerical results proving that a heavy-hole spin qubit can be reliably initialised, rotated and entangled by electrical means alone. This is due to strong Rashba-like spin–orbit interaction terms enabled by the interface inversion asymmetry. Single qubit rotations rely on electric-dipole spin resonance (EDSR), which is strongly enhanced by interface-induced spin–orbit terms. Entanglement can be accomplished by Coulomb exchange, coupling to a resonator, or spin–orbit induced dipole–dipole interactions. By analysing the qubit sensitivity to charge noise, we demonstrate that interface-induced spin–orbit terms are responsible for sweet spots in the dephasing time {T}2* as a function of the top gate electric field, which are close to maxima in the EDSR strength, where the EDSR gate has high fidelity. We show that both qubits can be described using the same starting Hamiltonian, and by comparing their properties we show that the complex interplay of bulk and interface-induced spin–orbit terms allows a high degree of electrical control and makes acceptors potential candidates for scalable quantum computation in Si.

  14. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    PubMed

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  15. Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin.

    PubMed Central

    Watanabe, Takeshi; Ariga, Yumiko; Sato, Urara; Toratani, Tadayuki; Hashimoto, Masayuki; Nikaidou, Naoki; Kezuka, Yuichiro; Nonaka, Takamasa; Sugiyama, Junji

    2003-01-01

    Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure. PMID:12930197

  16. The Klebsiella pneumoniae O12 ATP-binding Cassette (ABC) Transporter Recognizes the Terminal Residue of Its O-antigen Polysaccharide Substrate.

    PubMed

    Mann, Evan; Mallette, Evan; Clarke, Bradley R; Kimber, Matthew S; Whitfield, Chris

    2016-04-29

    Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a β-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal β-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures. PMID:26934919

  17. Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N-propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction†,‡

    PubMed Central

    Srivastava, Dhiraj; Zhu, Weidong; Johnson, William H.; Whitman, Christian P.; Becker, Donald F.; Tanner, John J.

    2013-01-01

    Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the ε-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline reduced, membrane-associated form. The 2.15 Å resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane binding domain include major reorganization of the flavin ribityl chain, severe (35 degree) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5), Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the β1-αl loop to complete the active site. PMID:19994913

  18. Optical activation behavior of ion implanted acceptor species in GaN

    SciTech Connect

    Skromme, B.J.; Martinez, G.L.

    2000-07-01

    Ion implantation is used to investigate the spectroscopic properties of Mg, Be, and C acceptors in GaN. Activation of these species is studied using low temperature photoluminescence (PL). Low dose implants into high quality undoped hydride vapor phase epitaxial (HVPE) material are used in conjunction with high temperature (1300 C) rapid thermal anneals to obtain high quality spectra. Dramatic, dose-dependent evidence of Mg acceptor activation is observed without any co-implants, including a strong, sharp neutral Mg acceptor-bound exciton and strong donor-acceptor pair peaks. Variable temperature measurements reveal a band-to-acceptor transition, whose energy yields an optical binding energy of 224 meV. Be and C implants yield only slight evidence of shallow acceptor-related features and produce dose-correlated 2.2 eV PL, attributed to residual implantation damage. Their poor optical activation is tentatively attributed to insufficient vacancy production by these lighter ions. Clear evidence is obtained for donor-Zn acceptor pair and acceptor-bound exciton peaks in Zn-doped HVPE material.

  19. The crystal structure of Pseudomonas avirulence protein AvrPphB: A papain-like fold with a distinct substrate binding site

    SciTech Connect

    Zhu, M.; Shao, F.; Innes, R.W.; Dixon, J.E.; Xu, Z.

    2010-03-08

    AvrPphB is an avirulence (Avr) protein from the plant pathogen Pseudomonas syringae that can trigger a disease-resistance response in a number of host plants including Arabidopsis. AvrPphB belongs to a novel family of cysteine proteases with the charter member of this family being the Yersinia effector protein YopT. AvrPphB has a very stringent substrate specificity, catalyzing a single proteolytic cleavage in the Arabidopsis serine/threonine kinase PBS1. We have determined the crystal structure of AvrPphB by x-ray crystallography at 1.35-{angstrom} resolution. The structure is composed of a central antiparallel {beta}-sheet, with {alpha}-helices packing on both sides of the sheet to form a two-lobe structure. The core of this structure resembles the papain-like cysteine proteases. The similarity includes the AvrPphB active site catalytic triad of Cys-98, His-212, and Asp-227 and the oxyanion hole residue Asn-93. Based on analogy with inhibitor complexes of the papain-like proteases, we propose a model for the substrate-binding mechanism of AvrPphB. A deep and positively charged pocket (S2) and a neighboring shallow surface (S3) likely bind to aspartic acid and glycine residues in the substrate located two (P2) and three (P3) residues N terminal to the cleavage site, respectively. Further implications about the specificity of plant pathogen recognition are also discussed.

  20. Identification of Phosphoinositide-Binding Protein PATELLIN2 as a Substrate of Arabidopsis MPK4 MAP Kinase during Septum Formation in Cytokinesis.

    PubMed

    Suzuki, Takamasa; Matsushima, Chiyuki; Nishimura, Shingo; Higashiyama, Tetsuya; Sasabe, Michiko; Machida, Yasunori

    2016-08-01

    The phosphorylation of proteins by protein kinases controls many cellular and physiological processes, which include intracellular signal transduction. However, the underlying molecular mechanisms of such controls and numerous substrates of protein kinases remain to be characterized. The mitogen-activated protein kinase (MAPK) cascade is of particular importance in a variety of extracellular and intracellular signaling processes. In plant cells, the progression of cytokinesis is an excellent example of an intracellular phenomenon that requires the MAPK cascade. However, the way in which MAPKs control downstream processes during cytokinesis in plant cells remains to be fully determined. We show here that comparisons, by two-dimensional difference gel electrophoresis, of phosphorylated proteins from wild-type Arabidopsis thaliana and mutant plants defective in a MAPK cascade allow identification of substrates of a specific MAPK. Using this method, we identified the PATELLIN2 (PATL2) protein, which has a SEC14 domain, as a substrate of MPK4 MAP kinase. PATL2 was concentrated at the cell division plane, as is MPK4, and had binding affinity for phosphoinositides. This binding affinity was altered after phosphorylation of PATL2 by MPK4, suggesting a role for the MAPK cascade in the formation of cell plates via regeneration of membranes during cytokinesis. PMID:27335345

  1. Origin of low sodium capacity in graphite and generally weak substrate binding of Na and Mg among alkali and alkaline earth metals

    NASA Astrophysics Data System (ADS)

    Liu, Yuanyue; Merinov, Boris V.; Goddard, William A., III

    2016-04-01

    It is well known that graphite has a low capacity for Na but a high capacity for other alkali metals. The growing interest in alternative cation batteries beyond Li makes it particularly important to elucidate the origin of this behavior, which is not well understood. In examining this question, we find a quite general phenomenon: among the alkali and alkaline earth metals, Na and Mg generally have the weakest chemical binding to a given substrate, compared with the other elements in the same column of the periodic table. We demonstrate this with quantum mechanics calculations for a wide range of substrate materials (not limited to C) covering a variety of structures and chemical compositions. The phenomenon arises from the competition between trends in the ionization energy and the ion-substrate coupling, down the columns of the periodic table. Consequently, the cathodic voltage for Na and Mg is expected to be lower than those for other metals in the same column. This generality provides a basis for analyzing the binding of alkali and alkaline earth metal atoms over a broad range of systems.

  2. Origin of low sodium capacity in graphite and generally weak substrate binding of Na and Mg among alkali and alkaline earth metals.

    PubMed

    Liu, Yuanyue; Merinov, Boris V; Goddard, William A

    2016-04-01

    It is well known that graphite has a low capacity for Na but a high capacity for other alkali metals. The growing interest in alternative cation batteries beyond Li makes it particularly important to elucidate the origin of this behavior, which is not well understood. In examining this question, we find a quite general phenomenon: among the alkali and alkaline earth metals, Na and Mg generally have the weakest chemical binding to a given substrate, compared with the other elements in the same column of the periodic table. We demonstrate this with quantum mechanics calculations for a wide range of substrate materials (not limited to C) covering a variety of structures and chemical compositions. The phenomenon arises from the competition between trends in the ionization energy and the ion-substrate coupling, down the columns of the periodic table. Consequently, the cathodic voltage for Na and Mg is expected to be lower than those for other metals in the same column. This generality provides a basis for analyzing the binding of alkali and alkaline earth metal atoms over a broad range of systems. PMID:27001855

  3. Identification of Phosphoinositide-Binding Protein PATELLIN2 as a Substrate of Arabidopsis MPK4 MAP Kinase during Septum Formation in Cytokinesis

    PubMed Central

    Suzuki, Takamasa; Matsushima, Chiyuki; Nishimura, Shingo; Higashiyama, Tetsuya; Sasabe, Michiko; Machida, Yasunori

    2016-01-01

    The phosphorylation of proteins by protein kinases controls many cellular and physiological processes, which include intracellular signal transduction. However, the underlying molecular mechanisms of such controls and numerous substrates of protein kinases remain to be characterized. The mitogen-activated protein kinase (MAPK) cascade is of particular importance in a variety of extracellular and intracellular signaling processes. In plant cells, the progression of cytokinesis is an excellent example of an intracellular phenomenon that requires the MAPK cascade. However, the way in which MAPKs control downstream processes during cytokinesis in plant cells remains to be fully determined. We show here that comparisons, by two-dimensional difference gel electrophoresis, of phosphorylated proteins from wild-type Arabidopsis thaliana and mutant plants defective in a MAPK cascade allow identification of substrates of a specific MAPK. Using this method, we identified the PATELLIN2 (PATL2) protein, which has a SEC14 domain, as a substrate of MPK4 MAP kinase. PATL2 was concentrated at the cell division plane, as is MPK4, and had binding affinity for phosphoinositides. This binding affinity was altered after phosphorylation of PATL2 by MPK4, suggesting a role for the MAPK cascade in the formation of cell plates via regeneration of membranes during cytokinesis. PMID:27335345

  4. Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)*

    PubMed Central

    Makkinje, Anthony; Vanden Borre, Pierre; Near, Richard I.; Patel, Prayag S.; Lerner, Adam

    2012-01-01

    The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. PMID:22711540

  5. The impact of molecular manipulation in residue 114 of Human Immunodeficiency Virus Type-1 reverse transcriptase on dNTP substrate binding and viral replication

    PubMed Central

    Van Cor-Hosmer, Sarah K.; Daddacha, Waaqo; Kelly, Z; Tsurumi, Amy; Kennedy, Edward M.; Kim, Baek

    2013-01-01

    Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has a unique tight binding to dNTP substrates. Structural modeling of Ala-114 of HIV-1 RT suggests that longer side chains at this residue can reduce the space normally occupied by the sugar moiety of an incoming dNTP. Indeed, mutations at Ala-114 decrease the ability of RT to synthesize DNA at low dNTP concentrations and reduce the dNTP-binding affinity (Kd) of RT. However, the Kd values of WT and A114C RT remained equivalent with an acylic dNTP substrate. Finally, mutant A114 RT HIV-1 vectors displayed a greatly reduced transduction in nondividing human lung fibroblasts (HLFs), while WT HIV-1 vector efficiently transduced both dividing and nondividing HLFs. Together these data support that the A114 residue of HIV-1 RT plays a key mechanistic role in the dNTP binding of HIV-1 RT and the unique viral infectivity of target cell types with low dNTP pools. PMID:22153297

  6. Anatomy of a conformational transition of beta-strand 6 in soybean beta-amylase caused by substrate (or inhibitor) binding to the catalytical site.

    PubMed Central

    Pujadas, G.; Palau, J.

    1997-01-01

    A computational study of the five soybean beta-amylase X-ray structure reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the beta-strand 6 (residues 341-343) in the (beta/alpha)8 molecular scaffold. Backbone distortions that involve considerable changes in the phi and psi angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA-layer (at the C-terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand-binding process, there is a negative enthalpy change for these residues (-27 and -34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed "stability-function" hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452-456). They are also in good agreement with other experimental results in the literature that describe the role of the 341-343 segment in beta-amylase activity. Site-directed mutagenesis focused on these residues could be useful for undertaking functional studies of beta-amylase. PMID:9385643

  7. TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

    PubMed

    Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

    2016-06-01

    Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26669798

  8. Insights from molecular dynamics: the binding site of cocaine in the dopamine transporter and permeation pathways of substrates in the leucine and dopamine transporters

    PubMed Central

    Merchant, Bonnie A.; Madura, Jeffry D.

    2012-01-01

    The dopamine transporter (DAT) facilitates the regulation of synaptic neurotransmitter levels. As a target for therapeutic and illicit psycho-stimulant drugs like antidepressants and cocaine, DAT has been studied intensively. Despite a wealth of mutational and physiological data regarding DAT, the structure remains unsolved and details of the transport mechanism, binding sites and conformational changes remain debated. A bacterial homologue of DAT, the leucine transporter (LeuTAa) has been used as a template and framework for modeling and understanding DAT. Free energy profiles obtained from Multi-Configuration Thermodynamic Integration allowed us to correctly identify the primary and secondary binding pockets of LeuTAa. A comparison of free energy profiles for dopamine and cocaine in DAT suggests that the binding site of cocaine is located in a secondary pocket, not the primary substrate site. Two recurring primary pathways for intracellular substrate release from the primary pocket are identified in both transporters using the Random Acceleration Molecular Dynamics method. One pathway appears to follow transmembranes (TMs) 1a and 6b while the other pathway follows along TMs 6b and 8. Interestingly, we observe that a single sodium ion is co-transported with leucine during both simulations types. PMID:23079638

  9. X-ray Structure and Molecular Dynamics Simulations of Endoglucanase 3 from Trichoderma harzianum: Structural Organization and Substrate Recognition by Endoglucanases That Lack Cellulose Binding Module

    PubMed Central

    Prates, Érica T.; Stankovic, Ivana; Silveira, Rodrigo L.; Liberato, Marcelo V.; Henrique-Silva, Flávio; Pereira, Nei; Polikarpov, Igor; Skaf, Munir S.

    2013-01-01

    Plant biomass holds a promise for the production of second-generation ethanol via enzymatic hydrolysis, but its utilization as a biofuel resource is currently limited to a large extent by the cost and low efficiency of the cellulolytic enzymes. Considerable efforts have been dedicated to elucidate the mechanisms of the enzymatic process. It is well known that most cellulases possess a catalytic core domain and a carbohydrate binding module (CBM), without which the enzymatic activity can be drastically reduced. However, Cel12A members of the glycosyl hydrolases family 12 (GHF12) do not bear a CBM and yet are able to hydrolyze amorphous cellulose quite efficiently. Here, we use X-ray crystallography and molecular dynamics simulations to unravel the molecular basis underlying the catalytic capability of endoglucanase 3 from Trichoderma harzianum (ThEG3), a member of the GHF12 enzymes that lacks a CBM. A comparative analysis with the Cellulomonas fimi CBM identifies important residues mediating interactions of EG3s with amorphous regions of the cellulose. For instance, three aromatic residues constitute a harboring wall of hydrophobic contacts with the substrate in both ThEG3 and CfCBM structures. Moreover, residues at the entrance of the active site cleft of ThEG3 are identified, which might hydrogen bond to the substrate. We advocate that the ThEG3 residues Asn152 and Glu201 interact with the substrate similarly to the corresponding CfCBM residues Asn81 and Arg75. Altogether, these results show that CBM motifs are incorporated within the ThEG3 catalytic domain and suggest that the enzymatic efficiency is associated with the length and position of the substrate chain, being higher when the substrate interact with the aromatic residues at the entrance of the cleft and the catalytic triad. Our results provide guidelines for rational protein engineering aiming to improve interactions of GHF12 enzymes with cellulosic substrates. PMID:23516599

  10. Changes in the catalytic properties of Pyrococcus furiosus thermostable amylase by mutagenesis of the substrate binding sites.

    PubMed

    Yang, Sung-Jae; Min, Byoung-Chul; Kim, Young-Wan; Jang, Sang-Mok; Lee, Byong-Hoon; Park, Kwan-Hwa

    2007-09-01

    Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced alpha-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over beta-CD, calculated by comparing k(cat)/K(m) ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes. PMID:17630303

  11. Model of the exofacial substrate-binding site and helical folding of the human Glut1 glucose transporter based on scanning mutagenesis.

    PubMed

    Mueckler, Mike; Makepeace, Carol

    2009-06-30

    Transmembrane helix 9 of the Glut1 glucose transporter was analyzed by cysteine-scanning mutagenesis and the substituted cysteine accessibility method (SCAM). A cysteine-less (C-less) template transporter containing amino acid substitutions for the six native cysteine residues present in human Glut1 was used to generate a series of 21 mutant transporters by substituting each successive residue in predicted transmembrane segment 9 with a cysteine residue. The mutant proteins were expressed in Xenopus oocytes, and their specific transport activities were directly compared to that of the parental C-less molecule whose function has been shown to be indistinguishable from that of native Glut1. Only a single mutant (G340C) had activity that was reduced (by 75%) relative to that of the C-less parent. These data suggest that none of the amino acid side chains in helix 9 is absolutely required for transport function and that this helix is not likely to be directly involved in substrate binding or translocation. Transport activity of the cysteine mutants was also tested after incubation of oocytes in the presence of the impermeant sulfhydryl-specific reagent, p-chloromercuribenzene sulfonate (pCMBS). Only a single mutant (T352C) exhibited transport inhibition in the presence of pCMBS, and the extent of inhibition was minimal (11%), indicating that only a very small portion of helix 9 is accessible to the external solvent. These results are consistent with the conclusion that helix 9 plays an outer stabilizing role for the inner helical bundle predicted to form the exofacial substrate-binding site. All 12 of the predicted transmembrane segments of Glut1 encompassing 252 amino acid residues and more than 50% of the complete polypeptide sequence have now been analyzed by scanning mutagenesis and SCAM. An updated model is presented for the outward-facing substrate-binding site and relative orientation of the 12 transmembrane helices of Glut1. PMID:19449892

  12. Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality.

    PubMed

    Lei, Zhen; Gao, Jiaxue; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-04-27

    We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL(-1) for MMP-2 and 5.7 pg mL(-1) for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained. PMID:27049528

  13. Use of a NICA-Donnan approach for analysis of proton binding to a lignocellulosic substrate extracted from wheat bran.

    PubMed

    Bouanda, J; Dupont, L; Dumonceau, J; Aplincourt, M

    2002-06-01

    The acid-base properties of a lignocellulosic substrate extracted from wheat bran have been investigated. The lignocellulosic substrate was first studied by use of FTIR, XPS, and solid-state 13C NMR to characterize the surface-active groups. Major contributions arise from the presence of carboxylic and phenolic sites. The former are associated with long-chain fatty acids and the latter are constituent units of lignin. All ionizable sites were quantified by use of the Ca-acetate method and by potentiometric titrations in non-aqueous media. Results were compared with those from conductimetric titrations in water and in the presence of barium ions. Protometric titration curves for the lignocellulosic substrate were obtained at several ionic strengths. Data were also treated with the NICA-Donnan model to determine the intrinsic ionization parameters. PMID:12043021

  14. An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling, inhibitory studies and molecular modeling.

    PubMed

    Węglarz-Tomczak, Ewelina; Poręba, Marcin; Byzia, Anna; Berlicki, Łukasz; Nocek, Bogusław; Mulligan, Rory; Joachimiak, Andrzej; Drąg, Marcin; Mucha, Artur

    2013-02-01

    Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria. To explore the possibility of NmAPN being a potential drug target for inhibition and development of novel therapeutic agents, the specificity of the S1 and S1' binding sites was explored using an integrated approach. Initially, an extensive library consisting of almost 100 fluorogenic substrates derived from both natural and unnatural amino acids, were used to obtain a detailed substrate fingerprint of the S1 pocket of NmAPN. A broad substrate tolerance of NmAPN was revealed, with bulky basic and hydrophobic ligands being the most favored substrates. Additionally, the potency of a set of organophosphorus inhibitors of neutral aminopeptidases, amino acid and dipeptide analogs was determined. Inhibition constants in the nanomolar range, determined for phosphinic dipeptides, proves the positive increase in inhibition impact of the P1' ligand elongation. The results were further verified via molecular modeling and docking of canonical aminopeptidase phosphinic dipeptide inhibitors in the NmAPN active site. These studies present comprehensive characterization of interactions responsible for specific ligand binding. This knowledge provides invaluable insight into understanding of the enzyme and development of novel NmAPN inhibitors. PMID:23131591

  15. The Aspergillus nidulans proline permease as a model for understanding the factors determining substrate binding and specificity of fungal amino acid transporters.

    PubMed

    Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky

    2015-03-01

    Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly(56), Thr(57)), TMS3 (Glu(138)), and TMS6 (Phe(248)), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393

  16. Crystal Structure of OXA-58 with the Substrate-Binding Cleft in a Closed State: Insights into the Mobility and Stability of the OXA-58 Structure

    PubMed Central

    Saino, Hiromichi; Sugiyabu, Tomohiro; Ueno, Go; Yamamoto, Masaki; Ishii, Yoshikazu; Miyano, Masashi

    2015-01-01

    OXA-58 is a class D β-lactamase from the multi-drug resistant Acinetobacter baumannii. We determined the crystal structure of OXA-58 in a novel crystal, and revealed the structure of the substrate-binding cleft in a closed state, distinct from a previously reported OXA-58 crystal structure with the binding cleft in an open state. In the closed state, the movement of three loops (α3–α4, β6–β7, and β8–α10) forms an arch-like architecture over the binding cleft through interaction between the Phe113 residues of α3–α4 and Met225 of β6–β7. This structure suggests the involvement of these flexible loops in OXA-58 substrate binding. In contrast to the mobile loops, the Ω-loop appeared static, including the conserved loop residues and their hydrogen bonds; the pivotal residue Trp169 within the Ω-loop, ζ-carbamic acid of the modified base catalyst residue Lys86, and nucleophilic residue Ser83. The stability of OXA-58 was enhanced concomitant with an increase in the hydrolytic activity catalyzed by NaHCO3-dependent ζ-carbamic acid formation, with an EC50 of 0.34 mM. The W169A mutant enzyme was significantly thermally unstable even in the presence of 100 mM NaHCO3, whereas the S83A mutant was stabilized with NaHCO3-dependent activation. The ζ-carbamic acid was shown to increase not only OXA-58 hydrolytic activity but also OXA-58 stability through the formation of a hydrogen bond network connected to the Ω-loop with Ser83 and Trp169. Thus, the static Ω-loop is important for OXA-58 stability, whereas the mobile loops of the substrate-binding cleft form the basis for accommodation of the various substituents of β-lactam backbone. PMID:26701320

  17. The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement

    PubMed Central

    Prats-Ejarque, Guillem; Arranz-Trullén, Javier; Blanco, Jose A.; Pulido, David; Nogués, M. Victòria; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His15, His122 and Gln14) and cationic surface-exposed residues (His36, His39, Arg66 and His67). Kinetic characterization, together with prediction of protein–nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5′ end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His36–His39 dyad that facilitates the polynucleotide substrate catalysis. PMID:27013146

  18. The Crystal Structure Analysis of Group B Streptococcus Sortase C1: A Model for the ;Lid; Movement upon Substrate Binding

    SciTech Connect

    Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu; Ton-That, Hung; Narayana, Sthanam V.L.

    2012-02-07

    A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible 'lid' anchored in the active site. However, the mechanistic details of the 'lid' displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its 'lid' mutant and proposed a role of the 'lid' as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.

  19. The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement.

    PubMed

    Prats-Ejarque, Guillem; Arranz-Trullén, Javier; Blanco, Jose A; Pulido, David; Nogués, M Victòria; Moussaoui, Mohammed; Boix, Ester

    2016-06-01

    Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His(15), His(122) and Gln(14)) and cationic surface-exposed residues (His(36), His(39), Arg(66) and His(67)). Kinetic characterization, together with prediction of protein-nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5' end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His(36)-His(39) dyad that facilitates the polynucleotide substrate catalysis. PMID:27013146

  20. The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

    PubMed Central

    Nørby, Jens G.; Esmann, Mikael

    1997-01-01

    The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3−, eosin2−, p-nitrophenylphosphate, and Vmax for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na2SO4 ≈ Na-acetate < NaNO3 < NaSCN < NaClO4. We treated the influence of NaCl and Na2SO4 on Kdiss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3− effect was compatible with competitive binding of NO3− and ADP in addition to the general I-effect. Kdiss for E·NO3 was ∼32 mM. Analysis of Vmax/Km for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme. PMID:9154904

  1. DONOR-ACCEPTOR INTERACTIONS OF NITROGEN*

    PubMed Central

    Kimura, J. E.; Szent-Györgyi, A.

    1969-01-01

    The nitrogen atoms of organic molecules readily enter into donor-acceptor interactions, giving off an electron from their lone pair. Under favorable conditions the acceptor can form free radicals. S and O atoms behave likewise but less intensely. PMID:4306047

  2. Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

    NASA Technical Reports Server (NTRS)

    Nealson, K. H.; Moser, D. P.; Saffarini, D. A.

    1995-01-01

    Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

  3. Glucansucrase acceptor reactions with D-mannose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The main acceptor product of glucansucrases with D-mannose has not previously been identified. We used glucansucrases that form water-insoluble a-D-glucans to produce increased yields of acceptor products from D-mannose, and identified the major product as 6-O-a-D-glucopyranosyl-D-mannose. Glucansuc...

  4. The impact of time-varying phosphorus doping on ZnMgO thin films and achievement of dominant acceptor-bound-exciton peak

    NASA Astrophysics Data System (ADS)

    Saha, S.; Nagar, S.; Gupta, S. K.; Chakrabarti, S.

    2014-03-01

    ZnO is a highly efficient and promising semiconductor material because of its large bandgap (3.37 eV) and exciton binding energy (60 meV). MgO also has a very high bandgap (7.8 eV), and the incorporation of Mg into ZnO can result in an alloy with a bandgap of more than 4 eV . We used plasma immersion ion implantation to dope phosphorus into Zn0.85Mg0.15O for achieving p-type ZnMgO. RF sputtering was used to deposit ZnMgO on a Si substrate. Phosphorus doping was conducted from 10 s to 70 s. Rapid thermal annealing of the samples was performed to remove any implantation defects. A highly dominant acceptor-bound-exciton peak was observed at 3.36 eV by photoluminescence measurements, which continued to dominate from low temperature to room temperature. Donor-bound acceptor and free-electron acceptor peaks were also observed at 3.24 eV and 3.28 eV, respectively.

  5. Diversity of sugar acceptor of glycosyltransferase 1 from Bacillus cereus and its application for glucoside synthesis.

    PubMed

    Chiu, Hsi-Ho; Shen, Mo-Yuan; Liu, Yuan-Ting; Fu, Yu-Lieh; Chiu, Yu-An; Chen, Ya-Huei; Huang, Chin-Ping; Li, Yaw-Kuen

    2016-05-01

    Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-β-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are β-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules. PMID:26795959

  6. Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

    PubMed

    Hoggett, J G; Kellett, G L

    1976-06-15

    A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits. PMID

  7. Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

    PubMed

    Perrera, Claudia; Colombo, Riccardo; Valsasina, Barbara; Carpinelli, Patrizia; Troiani, Sonia; Modugno, Michele; Gianellini, Laura; Cappella, Paolo; Isacchi, Antonella; Moll, Jurgen; Rusconi, Luisa

    2010-04-16

    Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis. PMID:20177074

  8. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    SciTech Connect

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-07-12

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate (Gpp(NH)p) on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed (/sup 32/P)ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-..cap alpha../sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-..cap alpha../sub i/ or anti-..cap alpha../sub 0/ antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed.

  9. Structures of Human Cyctochrome P450 2E1: Insights Into the Binding of Inhibitors And Both Small Molecular Weight And Fatty Acid Substrates

    SciTech Connect

    Porubsky, P.R.; Meneely, K.M.; Scott, E.E.

    2009-05-21

    Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates >70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 {angstrom} for an indazole complex and 2.6 {angstrom} for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr{sup 303} within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe{sup 478} aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved {sup 216}QXXNN{sup 220} residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed {omega}-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.

  10. Crystal Structure of Human Liver delta {4}-3-Ketosteroid 5 beta-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

    SciTech Connect

    Di Costanzo,L.; Drury, J.; Penning, T.; Christianson, D.

    2008-01-01

    AKR1D1 (steroid 5{beta}-reductase) reduces all 4-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an a,{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a 4-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90 bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human 4-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP+ at 1.79- and 1.35- Angstroms resolution (HEPES bound in the active site), NADP+ and cortisone at 1.90- Angstroms resolution, NADP+ and progesterone at 2.03- Angstroms resolution, and NADP+ and testosterone at 1.62- Angstroms resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP+. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr58 and Glu120. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

  11. The Mammalian Hsp40 ERdj3 Requires Its Hsp70 Interaction and Substrate-binding Properties to Complement Various Yeast Hsp40-dependent Functions*

    PubMed Central

    Vembar, Shruthi S.; Jin, Yi; Brodsky, Jeffrey L.; Hendershot, Linda M.

    2009-01-01

    Heat shock proteins of 70 kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are highly conserved chaperone pairs that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER), it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ER-lumenal JDP, ERdj3, was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation of unique Hsp70-JDP pairs, are critical to specify in vivo function. PMID:19748898

  12. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

    PubMed

    Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

  13. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras

    PubMed Central

    Warren, Jeremy G.; Lincoln, James E.; Kirkpatrick, Bruce C.

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

  14. Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase.

    PubMed

    Irwin, P L; Pfeffer, P E; Doner, L W; Sapers, G M; Brewster, J D; Nagahashi, G; Hicks, K B

    1994-03-18

    The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs. PMID:8194069

  15. Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.

    PubMed

    Bjelic, Sinisa; Aqvist, Johan

    2004-11-23

    The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment. PMID:15544322

  16. the active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions.

    PubMed

    Gloeckl, Sarina; Tyndall, Joel D A; Stansfield, Scott H; Timms, Peter; Huston, Wilhelmina M

    2012-01-01

    HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding. PMID:22353774

  17. Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding*

    PubMed Central

    Wang, An; Stout, C. David; Zhang, Qinghai; Johnson, Eric F.

    2015-01-01

    P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds. PMID:25555909

  18. Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site

    PubMed Central

    Singh, Harkewal; Arentson, Benjamin W.; Becker, Donald F.; Tanner, John J.

    2014-01-01

    Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of l-proline to l-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004-residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å-long tunnel that links the two active sites and six smaller tunnels that connect the main tunnel to the bulk medium. The locations of these tunnels and their responses to ligand binding and flavin reduction suggest hypotheses about how proline, water, and quinones enter the tunnel system and where l-glutamate exits. Kinetic measurements show that glutamate production from proline occurs without a lag phase, consistent with substrate channeling and implying that the observed tunnel is functionally relevant. Furthermore, the structure of reduced PutA complexed with menadione bisulfite reveals the elusive quinone-binding site. The benzoquinone binds within 4.0 Å of the flavin si face, consistent with direct electron transfer. The location of the quinone site implies that the concave surface of the PutA dimer approaches the membrane. Altogether, these results provide insight into how PutAs couple proline oxidation to quinone reduction. PMID:24550478

  19. P130Cas Src-Binding and Substrate Domains Have Distinct Roles in Sustaining Focal Adhesion Disassembly and Promoting Cell Migration

    PubMed Central

    Meenderink, Leslie M.; Ryzhova, Larisa M.; Donato, Dominique M.; Gochberg, Daniel F.; Kaverina, Irina; Hanks, Steven K.

    2010-01-01

    The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation. PMID:20976150

  20. Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site.

    PubMed

    Singh, Harkewal; Arentson, Benjamin W; Becker, Donald F; Tanner, John J

    2014-03-01

    Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004-residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å-long tunnel that links the two active sites and six smaller tunnels that connect the main tunnel to the bulk medium. The locations of these tunnels and their responses to ligand binding and flavin reduction suggest hypotheses about how proline, water, and quinones enter the tunnel system and where L-glutamate exits. Kinetic measurements show that glutamate production from proline occurs without a lag phase, consistent with substrate channeling and implying that the observed tunnel is functionally relevant. Furthermore, the structure of reduced PutA complexed with menadione bisulfite reveals the elusive quinone-binding site. The benzoquinone binds within 4.0 Å of the flavin si face, consistent with direct electron transfer. The location of the quinone site implies that the concave surface of the PutA dimer approaches the membrane. Altogether, these results provide insight into how PutAs couple proline oxidation to quinone reduction. PMID:24550478

  1. P-glycoprotein substrate binding domains are located at the transmembrane domain/transmembrane domain interfaces: a combined photoaffinity labeling-protein homology modeling approach.

    PubMed

    Pleban, Karin; Kopp, Stephan; Csaszar, Edina; Peer, Michael; Hrebicek, Thomas; Rizzi, Andreas; Ecker, Gerhard F; Chiba, Peter

    2005-02-01

    P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps. PMID:15509712

  2. Evidence for separate substrate binding sites for hydrogen peroxide and cumene hydroperoxide (CHP) in the oxidation of ethanol by catalase

    SciTech Connect

    DeMaster, E.G.; Nagasawa,ss H.T.

    1986-03-01

    The oxidation of ethanol by purified bovine liver catalase (Sigma, C-40) can be supported by H/sub 2/O/sub 2/ or by CHP. The time course of the H/sub 2/O/sub 2/ supported reaction (using glucose/glucose oxidase as the H/sub 2/O/sub 2/ source) was linear for at least one hr, whereas the rate of acetaldehyde formation in the CHP (4.2 mM) supported reaction decreased with time. When catalase was exposed o CHP for 5 min before the addition of ethanol, the rate of CHP supported ethanol oxidation was reduced by more than 90% compared to incubations where the addition of ethanol preceded that of CHP. In the CHP inhibited state, the peroxidative activity of catalase was not restored by further addition of CHP or ethanol; however, addition of fresh catalase yielded its expected activity. Significantly, the CHP inhibited enzyme was equally effective as the untreated enzyme in catalyzing (a) the oxidation of ethanol in the presence H/sub 2/O/sub 2/ supported peroxidative activity as well as catalytic activity by CHP inhibited catalase points to separate binding sites for H/sub 2/O/sub 2/ and CHP in this reaction. Alternatively, CHP may bind adjacent to a common peroxide active site, thereby sterically impeding the binding of CHP - but not of H/sub 2/O/sub 2/ - to this active site.

  3. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter - substrate binding protein complex

    PubMed Central

    Nguyen, Phong T.; Li, Qi Wen; Kadaba, Neena S.; Lai, Jeffrey Y.; Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ~40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system. PMID:25803078

  4. Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site*

    PubMed Central

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D.; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R.; Vaughan, Roxanne A.; Henry, L. Keith

    2014-01-01

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([125I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [125I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  5. Structure-Based Mutagenesis of the Substrate-Recognition Domain of Nrdp1/FLRF Identifies the Binding Site for the Receptor Tyrosine Kinase ErbB3

    SciTech Connect

    Bouyain,S.; Leahy, D.

    2007-01-01

    The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.

  6. Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

    PubMed

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R; Vaughan, Roxanne A; Henry, L Keith

    2014-10-24

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  7. The cooperative binding of phenylalanine to phenylalanine 4-monooxygenase studied by 1H-NMR paramagnetic relaxation. Changes in water accessibility to the iron at the active site upon substrate binding.

    PubMed

    Martínez, A; Olafsdottir, S; Flatmark, T

    1993-01-15

    The effect of the paramagnetic high-spin Fe(III) ion in phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) on the water proton longitudinal relaxation rate has been used to study the environment of the iron center. The relaxation rate was measured as a function of the concentration of enzyme, substrate (phenylalanine), inhibitor (noradrenaline) and activator (lysolecithin), as well as of the temperature (18-40 degrees C) and the external magnetic field strength (100-600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (tau c) of 4.2(+/- 0.5) x 10(-10) s was calculated for the enzyme-substrate complex, which most likely represents the electron spin relaxation rate (tau s) for Fe(III) (S = 5/2) in this complex. The relaxation rate was proportional to the concentration of enzyme (0.04-1 mM) both in the absence and presence of phenylalanine, but the paramagnetic molar relaxivity at 400 MHz and 22 degrees C decreased from 2.2(+/- 0.05) x 10(3) s-1.M-1 in the enzyme as isolated to 1.2(+/- 0.06) x 10(3) s-1.M-1 in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 +/- 0.8 kJ/mol to -1.5 +/- 0.2 kJ/mol upon incubation of the enzyme with 5 mM phenylalanine. The results obtained can be interpreted in terms of a slowly exchanging water molecule coordinated to the catalytic paramagnetic Fe(III) in the native and resting enzyme, and that this water molecule seems to be displaced from coordination on the binding of substrate or inhibitor. Moreover, the effect of increasing concentrations of phenylalanine and noradrenaline on the water proton relaxation rate and on the hydrophobic surface properties of the enzyme indicate that substrate and inhibitor induce a similar cooperative conformational change upon binding at the active site. By contrast, the activator lysolecithin does not seem to affect the interaction of water with the catalytic Fe

  8. Bright Solid-State Emission of Disilane-Bridged Donor-Acceptor-Donor and Acceptor-Donor-Acceptor Chromophores.

    PubMed

    Shimada, Masaki; Tsuchiya, Mizuho; Sakamoto, Ryota; Yamanoi, Yoshinori; Nishibori, Eiji; Sugimoto, Kunihisa; Nishihara, Hiroshi

    2016-02-01

    The development of disilane-bridged donor-acceptor-donor (D-Si-Si-A-Si-Si-D) and acceptor-donor-acceptor (A-Si-Si-D-Si-Si-A) compounds is described. Both types of compound showed strong emission (λem =ca. 500 and ca. 400 nm, respectively) in the solid state with high quantum yields (Φ: up to 0.85). Compound 4 exhibited aggregation-induced emission enhancement in solution. X-ray diffraction revealed that the crystal structures of 2, 4, and 12 had no intermolecular π-π interactions to suppress the nonradiative transition in the solid state. PMID:26822564

  9. Interface-induced heavy-hole/light-hole splitting of acceptors in silicon

    NASA Astrophysics Data System (ADS)

    Mol, J. A.; Salfi, J.; Rahman, R.; Hsueh, Y.; Miwa, J. A.; Klimeck, G.; Simmons, M. Y.; Rogge, S.

    2015-05-01

    The energy spectrum of spin-orbit coupled states of individual sub-surface boron acceptor dopants in silicon have been investigated using scanning tunneling spectroscopy at cryogenic temperatures. The spatially resolved tunnel spectra show two resonances, which we ascribe to the heavy- and light-hole Kramers doublets. This type of broken degeneracy has recently been argued to be advantageous for the lifetime of acceptor-based qubits [R. Ruskov and C. Tahan, Phys. Rev. B 88, 064308 (2013)]. The depth dependent energy splitting between the heavy- and light-hole Kramers doublets is consistent with tight binding calculations, and is in excess of 1 meV for all acceptors within the experimentally accessible depth range (<2 nm from the surface). These results will aid the development of tunable acceptor-based qubits in silicon with long coherence times and the possibility for electrical manipulation.

  10. Interface-induced heavy-hole/light-hole splitting of acceptors in silicon

    SciTech Connect

    Mol, J. A.; Salfi, J.; Simmons, M. Y.; Rogge, S.; Rahman, R.; Hsueh, Y.; Klimeck, G.; Miwa, J. A.

    2015-05-18

    The energy spectrum of spin-orbit coupled states of individual sub-surface boron acceptor dopants in silicon have been investigated using scanning tunneling spectroscopy at cryogenic temperatures. The spatially resolved tunnel spectra show two resonances, which we ascribe to the heavy- and light-hole Kramers doublets. This type of broken degeneracy has recently been argued to be advantageous for the lifetime of acceptor-based qubits [R. Ruskov and C. Tahan, Phys. Rev. B 88, 064308 (2013)]. The depth dependent energy splitting between the heavy- and light-hole Kramers doublets is consistent with tight binding calculations, and is in excess of 1 meV for all acceptors within the experimentally accessible depth range (<2 nm from the surface). These results will aid the development of tunable acceptor-based qubits in silicon with long coherence times and the possibility for electrical manipulation.

  11. Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B).

    PubMed

    Wright, David J; Tate, Christopher G

    2015-10-01

    The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4-5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. PMID:26143388

  12. Releasing the brakes in coagulation Factor IXa by co-operative maturation of the substrate-binding site.

    PubMed

    Kristensen, Line Hyltoft; Olsen, Ole H; Blouse, Grant E; Brandstetter, Hans

    2016-08-01

    Coagulation Factor IX is positioned at the merging point of the intrinsic and extrinsic blood coagulation cascades. Factor IXa (activated Factor IX) serves as the trigger for amplification of coagulation through formation of the so-called Xase complex, which is a ternary complex of Factor IXa, its substrate Factor X and the cofactor Factor VIIIa on the surface of activated platelets. Within the Xase complex the substrate turnover by Factor IXa is enhanced 200000-fold; however, the mechanistic and structural basis for this dramatic enhancement remains only partly understood. A multifaceted approach using enzymatic, biophysical and crystallographic methods to evaluate a key set of activity-enhanced Factor IXa variants has demonstrated a delicately balanced bidirectional network. Essential molecular interactions across multiple regions of the Factor IXa molecule co-operate in the maturation of the active site. This maturation is specifically facilitated by long-range communication through the Ile(212)-Ile(213) motif unique to Factor IXa and a flexibility of the 170-loop that is further dependent on the conformation in the Cys(168)-Cys(182) disulfide bond. Ultimately, the network consists of compensatory brakes (Val(16) and Ile(213)) and accelerators (Tyr(99) and Phe(174)) that together allow for a subtle fine-tuning of enzymatic activity. PMID:27208168

  13. Structure of Chlorobium tepidum sepiapterin reductase complex reveals the novel substrate binding mode for stereospecific production of L-threo-tetrahydrobiopterin.

    PubMed

    Supangat, Supangat; Seo, Kyung Hye; Choi, Yong Kee; Park, Young Shik; Son, Daeyoung; Han, Chang-deok; Lee, Kon Ho

    2006-01-27

    Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold. PMID:16308317

  14. Collagen-Gelatin Mixtures as Wound Model, and Substrates for VEGF-Mimetic Peptide Binding and Endothelial Cell Activation

    PubMed Central

    Chan, Tania R.; Stahl, Patrick J.; Li, Yang; Yu, S. Michael

    2015-01-01

    In humans, high level of collagen remodeling is seen during normal physiological events such as bone renewal, as well as in pathological conditions, such as arthritis, tumor growth and other chronic wounds. Our lab recently discovered that collagen mimetic peptide (CMP) is able to hybridize with denatured collagens at these collagen remodeling sites with high affinity. Here, we show that the CMP's high binding affinity to denatured collagens can be utilized to deliver angiogenic signals to scaffolds composed of heat-denatured collagens (gelatins). We first demonstrate hybridization between denatured collagens and QKCMP, a CMP with pro-angiogenic QK domain. We show that high levels of QKCMP can be immobilized to a new artificial matrix containing both fibrous type I collagen and heat denatured collagen through triple helix hybridization, and that the QKCMP is able to stimulate early angiogenic response of endothelial cells (ECs). We also show that the QKCMP can bind to excised tissues from burn injuries in cutaneous mouse model, suggesting its potential for promoting neovascularization of burn wounds. PMID:25584990

  15. Effect of cathode electron acceptors on simultaneous anaerobic sulfide and nitrate removal in microbial fuel cell.

    PubMed

    Cai, Jing; Zheng, Ping; Mahmood, Qaisar

    2016-01-01

    The current investigation reports the effect of cathode electron acceptors on simultaneous sulfide and nitrate removal in two-chamber microbial fuel cells (MFCs). Potassium permanganate and potassium ferricyanide were common cathode electron acceptors and evaluated for substrate removal and electricity generation. The abiotic MFCs produced electricity through spontaneous electrochemical oxidation of sulfide. In comparison with abiotic MFC, the biotic MFC showed better ability for simultaneous nitrate and sulfide removal along with electricity generation. Keeping external resistance of 1,000 Ω, both MFCs showed good capacities for substrate removal where nitrogen and sulfate were the main end products. The steady voltage with potassium permanganate electrodes was nearly twice that of with potassium ferricyanide. Cyclic voltammetry curves confirmed that the potassium permanganate had higher catalytic activity than potassium ferricyanide. The potassium permanganate may be a suitable choice as cathode electron acceptor for enhanced electricity generation during simultaneous treatment of sulfide and nitrate in MFCs. PMID:26901739

  16. Putative implication of alpha-amylase loop 7 in the mechanism of substrate binding and reaction products release.

    PubMed

    André, G; Tran, V

    2004-10-01

    Alpha-amylases are widespread endo-enzymes involved in the hydrolysis of internal alpha-(1,4) glycosidic linkages of starch polymers. Molecular modeling of amylose-amylase interactions is a step toward enzymatic mechanism understanding and rational design of new enzymes. From the crystallographic complex of barley alpha-amylase AMY2-acarbose, the static aspects of amylose-amylase docking have been characterized with a model of maltododecaose (DP12) (G. André, A. Buléon, R. Haser, and V. Tran, Biopolymers 1999, Vol. 50, pp. 751-762; G. André and V. Tran, Special Publication no. 246 1999, The Royal Society of Chemistry, H. J. Gilbert, G. J. Davies, B. Henrissat, and B. Svensson, Eds., Cambridge, pp. 165-174). These studies, consistent with the experimental subsite mapping (K. Bak-Jensen, G. André, V. Tran, and B. Svensson, Journal of Biological Chemistry, to be published), propose a propagation scheme for an amylose chain in the active cleft of AMY2. The topographical overview of alpha-amylases identified loop 7 as a conserved segment flanking the active site. Since some crystallographic experiments suspected its high flexibility, its putative motion was explored through a robotic scheme, an alternate route to dynamics simulations that consume CPU time. The present article describes the characteristics of the flexibility of loop 7: location and motion in AMY2. A back-and-forth motion with a large amplitude of more than 0.6 nm was evaluated. This movement could be triggered by two hinge residues. It results in the loop flipping over the active site to enhance the docking of the native helical substrate through specific interactions, it positions the catalytic residues, it distorts the substrate towards its transition state geometry, and finally monitors the release of the products after hydrolysis. The residues involved in the process are now rational mutation points in the hands of molecular biologists. PMID:15356864

  17. Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

    PubMed

    Sikorra, Stefan; Litschko, Christa; Müller, Carina; Thiel, Nadine; Galli, Thierry; Eichner, Timo; Binz, Thomas

    2016-01-29

    Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion. PMID:26523682

  18. The structure of barley alpha-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs.

    PubMed

    Robert, Xavier; Haser, Richard; Gottschalk, Tine E; Ratajczak, Fabien; Driguez, Hugues; Svensson, Birte; Aghajari, Nushin

    2003-08-01

    Though the three-dimensional structures of barley alpha-amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other alpha-amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in alpha-amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported. PMID:12906828

  19. Analysis of the Plastidic phosphate translocator Gene Family in Arabidopsis and Identification of New phosphate translocator-Homologous Transporters, Classified by Their Putative Substrate-Binding Site1

    PubMed Central

    Knappe, Silke; Flügge, Ulf-Ingo; Fischer, Karsten

    2003-01-01

    Analysis of the Arabidopsis genome revealed the complete set of plastidic phosphate translocator (pPT) genes. The Arabidopsis genome contains 16 pPT genes: single copies of genes coding for the triose phosphate/phosphate translocator and the xylulose phosphate/phosphate translocator, and two genes coding for each the phosphoenolpyruvate/phosphate translocator and the glucose-6-phosphate/phosphate translocator. A relatively high number of truncated phosphoenolpyruvate/phosphate translocator genes (six) and glucose-6-phosphate/phosphate translocator genes (four) could be detected with almost conserved intron/exon structures as compared with the functional genes. In addition, a variety of PT-homologous (PTh) genes could be identified in Arabidopsis and other organisms. They all belong to the drug/metabolite transporter superfamily showing significant similarities to nucleotide sugar transporters (NSTs). The pPT, PTh, and NST proteins all possess six to eight transmembrane helices. According to the analysis of conserved motifs in these proteins, the PTh proteins can be divided into (a) the lysine (Lys)/arginine group comprising only non-plant proteins, (b) the Lys-valine/alanine/glycine group of Arabidopsis proteins, (c) the Lys/asparagine group of Arabidopsis proteins, and (d) the Lys/threonine group of plant and non-plant proteins. None of these proteins have been characterized so far. The analysis of the putative substrate-binding sites of the pPT, PTh, and NST proteins led to the suggestion that all these proteins share common substrate-binding sites on either side of the membrane each of which contain a conserved Lys residue. PMID:12644669

  20. The evolution of substrate specificity-associated residues and Ca(2+) -binding motifs in EF-hand-containing type II NAD(P)H dehydrogenases.

    PubMed

    Hao, Meng-Shu; Rasmusson, Allan G

    2016-07-01

    Most eukaryotic organisms, except some animal clades, have mitochondrial alternative electron transport enzymes that allow respiration to bypass the energy coupling in oxidative phosphorylation. The energy bypass enzymes in plants include the external type II NAD(P)H dehydrogenases (DHs) of the NDB family, which are characterized by an EF-hand domain for Ca(2+) binding. Here we investigate these plant enzymes by combining molecular modeling with evolutionary analysis. Molecular modeling of the Arabidopsis thaliana AtNDB1 with the yeast ScNDI1 as template revealed distinct similarities in the core catalytic parts, and highlighted the interaction between the pyridine nucleotide and residues correlating with NAD(P)H substrate specificity. The EF-hand domain of AtNDB1 has no counterpart in ScNDI1, and was instead modeled with Ca(2+) -binding signal transducer proteins. Combined models displayed a proximity of the AtNDB1 EF-hand domain to the substrate entrance side of the catalytic part. Evolutionary analysis of the eukaryotic NDB-type proteins revealed ancient and recent reversions between the motif observed in proteins specific for NADH (acidic type) and NADPH (non-acidic type), and that the clade of enzymes with acidic motifs in angiosperms derives from non-acidic-motif NDB-type proteins present in basal plants, fungi and protists. The results suggest that Ca(2+) -dependent external NADPH oxidation is an ancient process, indicating that it has a fundamental importance for eukaryotic cellular redox metabolism. In contrast, the external NADH DHs in plants are products of a recent expansion, mirroring the expansion of the alternative oxidase family. PMID:27079180

  1. Access channels and methanol binding site to the CaMn4 cluster in Photosystem II based on solvent accessibility simulations, with implications for substrate water access.

    PubMed

    Ho, Felix M; Styring, Stenbjörn

    2008-02-01

    Given the tightly packed environment of Photosystem II (PSII), channels are expected to exist within the protein to allow the movement of small molecules to and from the oxygen evolving centre. In this report, we calculate solvent contact surfaces from the PSII crystal structures to identify such access channels for methanol and water molecules. In a previous study of the effects of methanol on the EPR split S1-, S3-, and S0-signals [Su et al. (2006) Biochemistry 45, 7617-7627], we proposed that methanol binds to one and the same Mn ion in all S-states. We find here that while channels of methanol dimensions were able to make contact with the CaMn4 cluster, only 3Mn and 4Mn were accessible to methanol. Combining this observation with spectroscopic data in the literature, we propose that 3Mn is the ion to which methanol binds. Furthermore, by calculating solvent contact surfaces for water, we found analogous and more extensive water accessible channels within PSII. On the basis of their structure, orientation, and electrostatic properties, we propose functional assignments of these channels as passages for substrate water access to the CaMn4 cluster, and for the exit of O2 and H+ that are released during water oxidation. Finally, we discuss the possible existence of a gating mechanism for the control of substrate water access to the CaMn4 cluster, based on the observation of a gap within the channel system that is formed by Ca2+ and several mechanistically very significant residues in the vicinity of the cluster. PMID:17964532

  2. Acceptor Products of Alternansucrase with Gentiobiose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. Previously, we showed that alternansucrase only forms...

  3. Synthetic CO.sub.2 acceptor

    DOEpatents

    Lancet, Michael S.; Curran, George P.

    1981-08-18

    A synthetic CO.sub.2 acceptor consisting essentially of at least one compound selected from the group consisting of calcium oxide and calcium carbonate supported in a refractory carrier matrix, the carrier having the general formula Ca.sub.5 (SiO.sub.4).sub.2 CO.sub.3. A method for producing the synthetic CO.sub.2 acceptor is also disclosed.

  4. Crystal Structure of Cytochrome P450 (CYP105P2) from Streptomyces peucetius and Its Conformational Changes in Response to Substrate Binding

    PubMed Central

    Lee, Chang Woo; Lee, Joo-Ho; Rimal, Hemraj; Park, Hyun; Lee, Jun Hyuck; Oh, Tae-Jin

    2016-01-01

    Cytochrome P450 monooxygenases (CYP, EC 1.14.14.1) belong to a large family of enzymes that catalyze the hydroxylation of various substrates. Here, we present the crystal structure of CYP105P2 isolated from Streptomyces peucetius ATCC27952 at a 2.1 Å resolution. The structure shows the presence of a pseudo-ligand molecule in the active site, which was co-purified fortuitously and is presumed to be a biphenyl derivative. Comparison with previously determined substrate-bound CYP structures showed that binding of the ligand produces large and distinctive conformational changes in α2–α3, α7–α9, and the C-terminal loop regions. This structural flexibility confirms our previous observation that CYP105P2 can accommodate a broad range of ligands. The structure complexed with a pseudo-ligand provides the first molecular view of CYP105P2–ligand interactions, and it indicates the involvement of hydrophobic residues (Pro82, Ala181, Met187, Leu189, Leu193, and Ile236) in the interactions between hydrophobic ligands and CYP105P2. These results provide useful insights into the structural changes involved in the recognition of different ligands by CYP105P2. PMID:27231902

  5. Structural basis for methyl-donor–dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD

    PubMed Central

    Ito, Takuhiro; Masuda, Isao; Yoshida, Ken-ichi; Goto-Ito, Sakurako; Sekine, Shun-ichi; Suh, Se Won; Hou, Ya-Ming; Yokoyama, Shigeyuki

    2015-01-01

    The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N1-methylguanosine (m1G) modification at position 37 in transfer RNAs (tRNAs) with the 36GG37 sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m1G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m1G37-tRNA. PMID:26183229

  6. Structural basis for methyl-donor-dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD.

    PubMed

    Ito, Takuhiro; Masuda, Isao; Yoshida, Ken-ichi; Goto-Ito, Sakurako; Sekine, Shun-ichi; Suh, Se Won; Hou, Ya-Ming; Yokoyama, Shigeyuki

    2015-08-01

    The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA. PMID:26183229

  7. Crystal Structure of Cytochrome P450 (CYP105P2) from Streptomyces peucetius and Its Conformational Changes in Response to Substrate Binding.

    PubMed

    Lee, Chang Woo; Lee, Joo-Ho; Rimal, Hemraj; Park, Hyun; Lee, Jun Hyuck; Oh, Tae-Jin

    2016-01-01

    Cytochrome P450 monooxygenases (CYP, EC 1.14.14.1) belong to a large family of enzymes that catalyze the hydroxylation of various substrates. Here, we present the crystal structure of CYP105P2 isolated from Streptomyces peucetius ATCC27952 at a 2.1 Å resolution. The structure shows the presence of a pseudo-ligand molecule in the active site, which was co-purified fortuitously and is presumed to be a biphenyl derivative. Comparison with previously determined substrate-bound CYP structures showed that binding of the ligand produces large and distinctive conformational changes in α2-α3, α7-α9, and the C-terminal loop regions. This structural flexibility confirms our previous observation that CYP105P2 can accommodate a broad range of ligands. The structure complexed with a pseudo-ligand provides the first molecular view of CYP105P2-ligand interactions, and it indicates the involvement of hydrophobic residues (Pro82, Ala181, Met187, Leu189, Leu193, and Ile236) in the interactions between hydrophobic ligands and CYP105P2. These results provide useful insights into the structural changes involved in the recognition of different ligands by CYP105P2. PMID:27231902

  8. Rearrangements of α-helical structures of FlgN chaperone control the binding affinity for its cognate substrates during flagellar type III export.

    PubMed

    Kinoshita, Miki; Nakanishi, Yuki; Furukawa, Yukio; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

    2016-08-01

    The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export. PMID:27178222

  9. Combining acid-base, redox and substrate binding functionalities to give a complete model for the [FeFe]-hydrogenase

    PubMed Central

    Camara, James M.; Rauchfuss, Thomas B.

    2012-01-01

    Some enzymes function by coupling substrate turnover with electron transfer from a redox cofactor such as ferredoxin. In the [FeFe]-hydrogenases, nature’s fastest catalysts for the production and oxidation of H2, the one-electron redox by a ferredoxin complements the one-electron redox by the diiron active site. In this Article, we replicate the function of the ferredoxins with the redox-active ligand Cp*Fe(C5Me4CH2PEt2) (FcP*). FcP* oxidizes at mild potentials, in contrast to most ferrocene-based ligands, which suggests that it might be a useful mimic of ferredoxin cofactors. The specific model is Fe2[(SCH2)2NBn](CO)3(FcP*)(dppv) (1), which contains the three functional components of the active site: a reactive diiron centre, an amine as a proton relay and, for the first time, a one-electron redox module. By virtue of the synthetic redox cofactor, [1]2+ exhibits unique reactivity towards hydrogen and CO. In the presence of excess oxidant and base, H2 oxidation by [1]2+ is catalytic. PMID:22169868

  10. The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis

    PubMed Central

    Otsuka, Taketo; Kirkham, Charmaine; Brauer, Aimee; Koszelak-Rosenblum, Mary; Malkowski, Michael G.

    2015-01-01

    Moraxella catarrhalis is an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to prevent M. catarrhalis infections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface of M. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement of M. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth of M. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen. PMID:26597985

  11. The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates*

    PubMed Central

    Gourlay, Keith; Hu, Jinguang; Arantes, Valdeir; Penttilä, Merja; Saddler, Jack N.

    2015-01-01

    Although the actions of many of the hydrolytic enzymes involved in cellulose hydrolysis are relatively well understood, the contributions that amorphogenesis-inducing proteins might contribute to cellulose deconstruction are still relatively undefined. Earlier work has shown that disruptive proteins, such as the non-hydrolytic non-oxidative protein Swollenin, can open up and disaggregate the less-ordered regions of lignocellulosic substrates. Within the cellulosic fraction, relatively disordered, amorphous regions known as dislocations are known to occur along the length of the fibers. It was postulated that Swollenin might act synergistically with hydrolytic enzymes to initiate biomass deconstruction within these dislocation regions. Carbohydrate binding modules (CBMs) that preferentially bind to cellulosic substructures were fluorescently labeled. They were imaged, using confocal microscopy, to assess the distribution of crystalline and amorphous cellulose at the fiber surface, as well as to track changes in surface morphology over the course of enzymatic hydrolysis and fiber fragmentation. Swollenin was shown to promote targeted disruption of the cellulosic structure at fiber dislocations. PMID:25527502

  12. Structural Studies of ROK Fructokinase YdhR from Bacillus subtilis: Insights into substrates binding and fructose specificity

    PubMed Central

    Nocek, B.; Stein, A.J.; Jedrzejczak, R.; Cuff, M. E.; Li, H.; Volkart, L.; Joachimiak, A.

    2011-01-01

    The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK) that phosphorylates d-fructose with ATP as a cofactor has been shown to be a member of this secondary system. Bioinformatics analysis has shown that FK is a member of the “ROK” (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/dfructose. All structures show that YdhR is a homo-dimer with a monomer composed of two similar α/βdomains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase. PMID:21185308

  13. Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5.

    PubMed

    Nemmara, Venkatesh V; Nicholas, Robert A; Pratt, R F

    2016-07-26

    Escherichia coli PBP5 (penicillin-binding protein 5) is a dd-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal d-alanyl-d-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB ), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the l-lysine of the original was replaced by l-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5. PMID:27420403

  14. The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.

    PubMed

    Metrick, Michael A; Temple, Joshua E; MacDonald, Gina

    2013-12-31

    The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions. PMID:24036048

  15. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    SciTech Connect

    Gao, Jinlan; Li, Xiaolu; Feng, Yue; Zhang, Bo; Miao, Shiying; Wang, Linfang; Wang, Na

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

  16. Structural studies of ROK fructokinase YdhR from Bacillus subtilis : insights into substrate binding and fructose specificity.

    SciTech Connect

    Nocek, B.; Stein, A.; Jedrzejczak, R.; Cuff, M.; Li, H.; Volkart, L.; Joachimiak, A.; Biosciences Division

    2011-02-18

    The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the 'ROK' (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar {alpha}/{beta} domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.

  17. Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study

    PubMed Central

    Gupta, Saurabh; Rao, Atmakuri Ramakrishna; Varadwaj, Pritish Kumar; De, Sachinandan; Mohapatra, Trilochan

    2015-01-01

    Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel. PMID:26313938

  18. Crystal Structure of Human Liver [delta][superscript 4]-3-Ketosteroid 5[beta]-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

    SciTech Connect

    Di Costanzo, Luigi; Drury, Jason E.; Penning, Trevor M.; Christianson, David W.

    2008-07-15

    AKR1D1 (steroid 5{beta}-reductase) reduces all {Delta}{sup 4}-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an {alpha}{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a {Delta}{sup 4}-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90{sup o} bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human {Delta}{sup 4}-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP{sup +} at 1.79- and 1.35-{angstrom} resolution (HEPES bound in the active site), NADP{sup +} and cortisone at 1.90-{angstrom} resolution, NADP{sup +} and progesterone at 2.03-{angstrom} resolution, and NADP{sup +} and testosterone at 1.62-{angstrom} resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP{sup +}. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr{sup 58} and Glu{sup 120}. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

  19. sup 17 O, sup 1 H, and sup 2 H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the (4Fe-4S) sup + cluster of aconitase

    SciTech Connect

    Werst, M.M.; Hoffman, B.M. ); Kennedy, M.C.; Beinert, H. )

    1990-11-01

    {sup 17}O electron nuclear double resonance (ENDOR) studies at X-band (9-GHz) and Q-band (35-GHz) microwave frequencies reveal that the (4Fe-4S){sup {plus}} cluster of substrate-free aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) binds solvent, H{sub x}O (x = 1,2). Previous {sup 17}O ENDOR studies had disclosed that H{sub x}{sup 17}O binds to the enzyme-substrate complex and also to complexes of enzyme with the substrate analogues trans-aconitate and nitroisocitrate (1-hydroxy-2-nitro-1,3-propanedicarboxylate). The authors have used {sup 1}H and {sup 2}H ENDOR to characterize these solvent species. The authors propose that the fourth ligand of Fe{sub a} in substrate-free enzyme is a hydroxyl ion from the solvent; upon binding of substrate or substrate analogues at this Fe{sub a} site, the solvent species becomes protonated to form a water molecule. Previous {sup 17}O and {sup 13}C ENDOR studies showed that only a single carboxyl, at C-2 of the propane backbone of cis-aconitate or at C-1 of the inhibitor nitroisocitrate, coordinates to the cluster. Together, these results imply that enzyme-catalyzed interconversion of citrate and isocitrate does not involve displacement of an endogenous fourth ligand, but rather addition of the anionic carboxylate ligand and a change in protonation state of a solvent species bound to Fe{sub a}. The authors further report the {sup 17}O hyperfine tensor parameters of the C-2 carboxyl oxygen of substrate bound to the cluster as determined by the field dependence of the {sup 17}O ENDOR signals. {sup 17}O ENDOR studies also show that the carboxyl group of the inhibitor trans-aconitate binds similarly to that off substrate.

  20. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    SciTech Connect

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  1. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    PubMed

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  2. Electronic structure of sub-surface Boron acceptors in silicon for potential qubits

    NASA Astrophysics Data System (ADS)

    Rahman, Rajib; Mol, Jan; Klimeck, Gerhard; Rogge, Sven

    2013-03-01

    Single acceptors in silicon are investigated as potential qubits. Due to the p-type nature of the valence band (VB), the acceptor states are less susceptible to the hyperfine interaction of the neighboring nuclear spins. The presence of a stronger spin-orbit coupling in the VB also enables the possibility of an all-electric qubit control. Whereas donor qubits exhibit exchange oscillation with separation distance due to conduction band valleys, Boron acceptors are expected to have smoother exchange curves. We investigate the electronic structure of single Boron acceptors in silicon in the presence of electric field, strain, magnetic field, and interfaces. Bulk Boron acceptors have a four-fold degenerate ground state 45 meV above the VB with angular momentum states of 3/2 and 1/2. An interface splits this manifold into Kramer's doublets. Application of E and B fields allow several possibilities for forming a two-level qubit driven by an ac electric field. We compare calculations from atomistic tight-binding theory to scanning tunneling microscope (STM) measurements and k.p calculations. The tight-binding method captures additional wavefunction symmetries due to the crystal that help to explain the STM measurements.

  3. Carbon Nanotube Patterning on a Metal Substrate

    NASA Technical Reports Server (NTRS)

    Nguyen, Cattien V. (Inventor)

    2016-01-01

    A CNT electron source, a method of manufacturing a CNT electron source, and a solar cell utilizing a CNT patterned sculptured substrate are disclosed. Embodiments utilize a metal substrate which enables CNTs to be grown directly from the substrate. An inhibitor may be applied to the metal substrate to inhibit growth of CNTs from the metal substrate. The inhibitor may be precisely applied to the metal substrate in any pattern, thereby enabling the positioning of the CNT groupings to be more precisely controlled. The surface roughness of the metal substrate may be varied to control the density of the CNTs within each CNT grouping. Further, an absorber layer and an acceptor layer may be applied to the CNT electron source to form a solar cell, where a voltage potential may be generated between the acceptor layer and the metal substrate in response to sunlight exposure.

  4. Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures

    PubMed Central

    Holliday, Michael J.; Camilloni, Carlo; Armstrong, Geoffrey S.; Isern, Nancy G.; Zhang, Fengli; Vendruscolo, Michele; Eisenmesser, Elan Z.

    2015-01-01

    Thermophilic proteins have found extensive use in research and industrial applications because of their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, constituting a ubiquitously expressed family of peptidyl–prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamic motions over several time scales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures to compare its structure, dynamics, and function to those of a mesophilic counterpart, human cyclophilin A (CypA). Unlike those of most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to that of CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Fast time scale (pico- to nanosecond) dynamics are largely conserved between the two proteins, in accordance with the high degree of structural similarity, although differences do exist in their temperature dependencies. Slower (microsecond) time scale motions are likewise localized to similar regions in the two proteins with some variability in their magnitudes yet

  5. Structure and dynamics of GeoCyp: a thermophilic cyclophilin with a novel substrate binding mechanism that functions efficiently at low temperatures

    SciTech Connect

    Holliday, Michael; Camilloni, Carlo; Armstrong, Geoffrey S.; Isern, Nancy G.; Zhang, Fengli; Vendruscolo, Michele; Eisenmesser, Elan Z.

    2015-05-26

    Thermophilic proteins have found extensive use in research and industrial applications due to their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, a ubiquitously expressed family of peptidyl-prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamic motions over several timescales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures in order to compare structure, dynamics, and function to a mesophilic counterpart, human Cyclophilin A (CypA). Unlike most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Despite subtle differences in conformational movements, dynamics over fast (ps-ns) and slow (μs) timescales are largely conserved between the two proteins.

  6. Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and -5/-6 of barley alpha-amylase 1.

    PubMed

    Mori, H; Bak-Jensen, K S; Gottschalk, T E; Motawia, M S; Damager, I; Møller, B L; Svensson, B

    2001-12-01

    Enzymatic properties of barley alpha-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95-->Ala/Thr) and +1/+2 (Met298-->Ala/Asn/Ser) as well as in the double mutants, Cys95-->Ala/Met298-->Ala/Asn/Ser. Cys95-->Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95-->Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95-->Ala and wild-type. Met298-->Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are < or = 30% and < or = 10% of wild-type, respectively. The activity of Cys95-->Ala/Met298-->Ala/Asn/Ser is 100-180% towards starch, and the kcat/Km is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95-->Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6"'-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3. PMID:11737209

  7. Doping of germanium and silicon crystals with non-hydrogenic acceptors for far infrared lasers

    DOEpatents

    Haller, Eugene E.; Brundermann, Erik

    2000-01-01

    A method for doping semiconductors used for far infrared lasers with non-hydrogenic acceptors having binding energies larger than the energy of the laser photons. Doping of germanium or silicon crystals with beryllium, zinc or copper. A far infrared laser comprising germanium crystals doped with double or triple acceptor dopants permitting the doped laser to be tuned continuously from 1 to 4 terahertz and to operate in continuous mode. A method for operating semiconductor hole population inversion lasers with a closed cycle refrigerator.

  8. Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication.

    PubMed

    Meadows, Corey W; Tsang, Jonathan E; Klinman, Judith P

    2014-10-22

    Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements, and the double mutant (Y25A:V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. Collisional quenching data at Trp87 further show a structural change in the active-site environment/solvation for V260A. In the aggregate, the temperature dependencies of the fluorescence data are distinct from the breaks in behavior previously reported for catalysis and hydrogen/deuterium exchange, attributed to time scales for the interconversion of protein conformational substates that are slower and more global than the local motions monitored within. An extended network of dynamical communication between the protein dimer surface and substrate- and cofactor-binding domains emerges from the flourescent data. PMID:25314615

  9. Structure and function of residue 104 and water molecules in the xenobiotic substrate-binding site in human glutathione S-transferase P1-1.

    PubMed

    Ji, X; Blaszczyk, J; Xiao, B; O'Donnell, R; Hu, X; Herzog, C; Singh, S V; Zimniak, P

    1999-08-10

    Two variants of human class pi glutathione (GSH) S-transferase 1-1 with either isoleucine or valine in position 104 (hGSTP1-1[I104] and hGSTP1-1[V104]) have distinct activity toward (+)-anti-7, 8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. To elucidate their structure-function relationship, we determined the crystal structures of the two variants in complex with GSBpd, the GSH conjugate of (+)-anti-BPDE, at 2.1 and 2.0 A resolution, respectively. The crystal structures reveal that residue 104 in the xenobiotic substrate-binding site (H-site) dictates the binding modes of the product molecule GSBpd with the following three consequences. First, the distance between the hydroxyl group of Y7 and the sulfur atom of GSBpd is 5.9 A in the hGSTP1-1[I104].GSBpd complex versus 3.2 A in the V104 variant. Second, one of the hydroxyl groups of GSBpd forms a direct hydrogen bond with R13 in hGSTP1-1[V104].GSBpd; in contrast, this hydrogen bond is not observed in the I104 complex. Third, in the hydrophilic portion of the H-site of the I104 complex, five H-site water molecules [Ji, X., et al. (1997) Biochemistry 36, 9690-9702] are observed, whereas in the V104 complex, two of the five have been displaced by the Bpd moiety of GSBpd. Although there is no direct hydrogen bond between Y108 (OH) and the hydroxyl groups of GSBpd, indirect hydrogen bonds mediated by water molecules are observed in both complexes, supporting the previously suggested role of the hydroxyl group of Y108 as an electrophilic participant in the addition of GSH to epoxides. PMID:10441116

  10. Carbon and group II acceptor coimplantation in GaAs

    SciTech Connect

    Morton, R.; Lau, S.S.; Poker, D.B.; Chu, P.K.; Fung, K.K.; Wang, N.

    1998-11-01

    Coimplantations of carbon and one of the group II acceptors, Mg, Zn, or Cd, were performed and compared to implantations involving only a single element (Mg, Zn, or Cd) or Ga and C coimplanted into GaAs substrates. The group II and C (II/C) coimplantations act to balance the crystal stoichiometry since group II atoms prefer to reside in the Ga sublattice and C prefers to reside in the As sublattice. The electrical characteristics of the various implantations were obtained from sheet and differential Hall measurements. Rutherford backscattering spectrometry was employed to determine the amount of implantation-induced damage which was then correlated to the amount of C activation in the group II/C coimplanted samples. It was found that coimplantation of the heavier group II acceptors, Zn and Cd, resulted in layers with larger peak hole concentrations. This is a result of the large amount of lattice damage created by these elements which is thought to provide the necessary abundance of As vacancies for C activation. Secondary ion mass spectroscopy measurements of the samples after implant activation indicate that C coimplantation significantly reduces the diffusivity of the group II acceptors. Cross-sectional transmission electron microscopy indicated a unique defect structure (extrinsic dislocation loops) for the cases of group II/C coimplantation. These dislocation loops are located at the diffusion front of the group II element in the samples and therefore have a rather profound influence on the diffusion of the group II elements. A rationalization of the defect structure and the effect it has on the diffusion of group II elements is given. {copyright} {ital 1998 American Institute of Physics.}

  11. Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes.

    PubMed

    Kreutzberger, Alex J B; Liang, Binyong; Kiessling, Volker; Tamm, Lukas K

    2016-05-24

    Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49-96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion. PMID:27178662

  12. Towards building artificial light harvesting complexes: enhanced singlet-singlet energy transfer between donor and acceptor pairs bound to albumins.

    PubMed

    Kumar, Challa V; Duff, Michael R

    2008-12-01

    Specific donor and acceptor pairs have been assembled in bovine serum albumin (BSA), at neutral pH and room temperature, and these dye-protein complexes indicated efficient donor to acceptor singlet-singlet energy transfer. For example, pyrene-1-butyric acid served as the donor and Coumarin 540A served as the acceptor. Both the donor and the acceptor bind to BSA with affinity constants in excess of 2x10(5) M(-1), as measured in absorption and circular dichroism (CD) spectral titrations. Simultaneous binding of both the donor and the acceptor chromophores was supported by CD spectra and one chromophore did not displace the other from the protein host, even when limited concentrations of the host were used. For example, a 1:1:1 complex between the donor, acceptor and the host can be readily formed, and spectral data clearly show that the binding sites are mutually exclusive. The ternary complexes (two different ligands bound to the same protein molecule) provided opportunities to examine singlet-singlet energy transfer between the protein-bound chromophores. Donor emission was quenched by the addition of the acceptor, in the presence of limited amounts of BSA, while no energy transfer was observed in the absence of the protein host, under the same conditions. The excitation spectra of the donor-acceptor-host complexes clearly show the sensitization of acceptor emission by the donor. Protein denaturation, as induced by the addition of urea or increasing the temperature to 360 K, inhibited energy transfer, which indicate that protein structure plays an important role. Sensitization also proceeded at low temperature (77 K) and diffusion of the donor or the acceptor is not required for energy transfer. Stern-Volmer quenching plots show that the quenching constant is (3.1+/-0.2)x10(4) M(-1), at low acceptor concentrations (<35 microM). Other albumins such as human and porcine proteins also served as good hosts for the above experiments. For the first time, non

  13. Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-α protein (PMPCA) cause a severe mitochondrial disease

    PubMed Central

    Joshi, Mugdha; Anselm, Irina; Shi, Jiahai; Bale, Tejus A.; Towne, Meghan; Schmitz-Abe, Klaus; Crowley, Laura; Giani, Felix C.; Kazerounian, Shideh; Markianos, Kyriacos; Lidov, Hart G.; Folkerth, Rebecca; Sankaran, Vijay G.; Agrawal, Pankaj B.

    2016-01-01

    We describe a large Lebanese family with two affected members, a young female proband and her male cousin, who had multisystem involvement including profound global developmental delay, severe hypotonia and weakness, respiratory insufficiency, blindness, and lactic acidemia—findings consistent with an underlying mitochondrial disorder. Whole-exome sequencing was performed on DNA from the proband and both parents. The proband and her cousin carried compound heterozygous mutations in the PMPCA gene that encodes for α-mitochondrial processing peptidase (α-MPP), a protein likely involved in the processing of mitochondrial proteins. The variants were located close to and postulated to affect the substrate binding glycine-rich loop of the α-MPP protein. Functional assays including immunofluorescence and western blot analysis on patient's fibroblasts revealed that these variants reduced α-MPP levels and impaired frataxin production and processing. We further determined that those defects could be rescued through the expression of exogenous wild-type PMPCA cDNA. Our findings link defective α-MPP protein to a severe mitochondrial disease. PMID:27148589

  14. Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-α protein (PMPCA) cause a severe mitochondrial disease.

    PubMed

    Joshi, Mugdha; Anselm, Irina; Shi, Jiahai; Bale, Tejus A; Towne, Meghan; Schmitz-Abe, Klaus; Crowley, Laura; Giani, Felix C; Kazerounian, Shideh; Markianos, Kyriacos; Lidov, Hart G; Folkerth, Rebecca; Sankaran, Vijay G; Agrawal, Pankaj B

    2016-05-01

    We describe a large Lebanese family with two affected members, a young female proband and her male cousin, who had multisystem involvement including profound global developmental delay, severe hypotonia and weakness, respiratory insufficiency, blindness, and lactic acidemia-findings consistent with an underlying mitochondrial disorder. Whole-exome sequencing was performed on DNA from the proband and both parents. The proband and her cousin carried compound heterozygous mutations in the PMPCA gene that encodes for α-mitochondrial processing peptidase (α-MPP), a protein likely involved in the processing of mitochondrial proteins. The variants were located close to and postulated to affect the substrate binding glycine-rich loop of the α-MPP protein. Functional assays including immunofluorescence and western blot analysis on patient's fibroblasts revealed that these variants reduced α-MPP levels and impaired frataxin production and processing. We further determined that those defects could be rescued through the expression of exogenous wild-type PMPCA cDNA. Our findings link defective α-MPP protein to a severe mitochondrial disease. PMID:27148589

  15. The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans.

    PubMed

    Nisha, M; Satyanarayana, T

    2015-07-01

    In order to understand the role of N1 domain (1-257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir-Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability. PMID:25573470

  16. Influence of the local environment on Mn acceptors in GaAs.

    PubMed

    Lee, Donghun; Gohlke, David; Benjamin, Anne; Gupta, Jay A

    2015-04-22

    As transistors continue to shrink toward nanoscale dimensions, their characteristics are increasingly dependent on the statistical variations of impurities in the semiconductor material. The scanning tunneling microscope (STM) can be used to not only study prototype devices with atomically precise placement of impurity atoms, but can also probe how the properties of these impurities depend on the local environment. Tunneling spectroscopy of Mn acceptors in GaAs indicates that surface-layer Mn act as a deep acceptor, with a hole binding energy that can be tuned by positioning charged defects nearby. Band bending induced by the tip or by these defects can also tune the ionization state of the acceptor complex, evident as a ring-like contrast in STM images. The interplay of these effects is explored over a wide range of defect distances, and understood using iterative simulations of tip-induced band bending. PMID:25782688

  17. Donor-Acceptor Heterojunction Configurations Based on DNA-Multichromophore Arrays.

    PubMed

    Nakamura, Mitsunobu; Tsuto, Koji; Jomura, Ayumi; Takada, Tadao; Yamana, Kazushige

    2015-08-10

    Multichromophore arrays of bis(2-thienyl)diketopyrrolopyrrole (DPP) and naphthalenediimide (NDI) with two Zn(II) -cyclens were constructed using thymidine DNA as a scaffold through the binding of the Zn(II) -cyclens with thymine bases. We demonstrate photocurrent generation in a donor-acceptor heterojunction configuration consisting of the DPP (donor) and NDI (acceptor) arrays co-immobilized on an Au electrode. The co-immobilized electrode exhibited good photocurrent responses because of the efficient charge separation between the DPP and NDI arrays. In contrast, an immobilized electrode consisting of randomly assembled DPP-NDI arrays generated no photocurrent response because DPP formed ground-state charge-transfer complexes with NDI in the randomly assembled arrays. Therefore, our approach to generate donor-acceptor heterojunctions based on DNA-multichromophore arrays is a useful method to efficiently generate photocurrent. PMID:26179473

  18. Electron Donor Acceptor Interactions. Final Progress Report

    SciTech Connect

    2002-08-16

    The Gordon Research Conference (GRC) on Electron Donor Acceptor Interactions was held at Salve Regina University, Newport, Rhode Island, 8/11-16/02. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  19. Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors.

    PubMed Central

    Frébortová, Jitka; Fraaije, Marco W; Galuszka, Petr; Sebela, Marek; Pec, Pavel; Hrbác, Jan; Novák, Ondrej; Bilyeu, Kristin D; English, James T; Frébort, Ivo

    2004-01-01

    The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s(-1) could be obtained. This suggests that the natural electron acceptor of the enzyme is quite probably a p-quinone or similar compound. By using the stopped-flow technique, it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (k(red)=950 s(-1)). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result, the enzyme displays a dual catalytic mode for oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors. PMID:14965342

  20. The true hydrophobicity of microsomal cytochrome P-450 in the rat: size dependence of the free energy of binding of a series of hydrocarbon substrates from the aqueous phase to the enzyme and to the membrane as derived from spectral binding data

    SciTech Connect

    Backes, W.L.; Hogaboom, M.; Canady, W.J.

    1982-04-25

    The associations of some hydrophobic substrates with cytochrome P-450 and with the microsomal membrane were studied. A series of aromatic hydrocarbons of increasing molecular size were used as a probe of the type I site of cytochrome P-450. The size dependence of the free energy (..delta..G/sup 0/) of binding was compared with the size dependence of transfer of the same compounds between the bulk solvent octanol and water. Similar size dependences were observed for the partition process (-0.68 kcal/mol/methylene group) and the binding of hydrocarbons to the type I binding site of cytochrome P-450 in both phenobarbital treated (-0.77 kcal/mol/methylene group) and untreated (-0.63 kcal/mol/methylene group) male rats. This is in contrast to earlier studies which did not take the dependence of the apparent binding constant upon enzyme concentration into account. Microsomal partitioning of the hydrocarbon substrates was quite different from the partitioning of those substrates between water and octanol. The size dependence of microsomal partitioning was found to be -0.27 kcal/mol/methylene group and -0.29 kcal/mol/methylene group in microsomes from untreated and phenobarbital-treated rats, respectively, indicating that the distribution of a substrate between water and a bulk organic solvent such as octanol is not a suitable model for an hepatic microsomal membrane, but that in certain ways it is an adequate model for enzyme-substrate complex formation. (JMT)

  1. Synthetic protease substrate n-benzoyl-L-argininyl-p-nitroanilide activates specific binding of (/sup 3/H)estradiol to a protein in rat pancreas: relationship of structure to activity

    SciTech Connect

    Grossman, A.

    1984-11-26

    N-benzoyl-L-argininyl-p-nitroanilide (BAN), a synthetic substrate for trypsin-like proteolytic enzymes, is a potent activator of (/sup 3/H)estradiol-binding to a protein present in rat pancreas. When partially purified, this protein is almost devoid of (/sup 3/H)estradiol-binding activity in the absence of an endogenous accessory factor. BAN can mimic the natural coligand in this steroid binding reaction. The effect of BAN is specific since a number of derivatives of this substance are inactive or may even inhibit steroid binding. It is unlikely that BAN exerts this stimulatory action indirectly, possibly by preventing proteolytic inactivation of the (/sup 3/H)estradiol-binding protein, since preincubation of the protein in the absence of BAN resulted neither in reduced rate, nor extent, of steroid binding following BAN addition. Also, a number of protease inhibitors had no effect on the binding reaction. Of those inhibitors tested, only antipain significantly enhanced binding of (/sup 3/H)estradiol, but only about 20 percent as effectively as BAN. 13 references, 1 figure, 2 tables.

  2. Theoretical calculation of the miniband-to-acceptor magnetoluminescence of semiconductor superlattices

    NASA Astrophysics Data System (ADS)

    Latgé, A.; Porras-Montenegro, N.; de Dios-Leyva, M.; Oliveira, L. E.

    1997-05-01

    The acceptor-related photoluminescence of a GaAs-(Ga,Al)As superlattice, under the influence of a magnetic field applied parallel to the interfaces, is theoretically studied following a variational procedure within the effective-mass approximation. Electron and hole magnetic Landau levels and envelope wave functions were obtained by an expansion in terms of sine functions, whereas for the impurity levels the envelope functions were taken as products of sine and hydrogenic-like variational functions. Impurity binding energies and wave functions are obtained for acceptors at a general position in the superlattice and for different in-plane magnetic fields. Theoretical results corresponding to transitions from the conduction subband to states of acceptors (miniband-to-acceptor e-A0 transitions) at the edge and center positions of the GaAs quantum well compare well with available experimental data by Skromme et al. [Phys. Rev. Lett. 65, 2050 (1990)] on the magnetic-field dependence of the photoluminescence peak position of conduction miniband-to-acceptor transitions for different temperatures and values of the superlattice period.

  3. X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues.

    PubMed

    Gordon, Roni D; Sivarajah, Prashanth; Satkunarajah, Malathy; Ma, Dengbo; Tarling, Chris A; Vizitiu, Dragos; Withers, Stephen G; Rini, James M

    2006-06-30

    The Golgi-resident glycosyltransferase, UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I), initiates the conversion of high-mannose oligosaccharides to complex and hybrid structures in the biosynthesis of N-linked glycans. Reported here are the X-ray crystal structures of GnT I in complex with UDP-CH2-GlcNAc (a non-hydrolyzable C-glycosidic phosphonate), UDP-2-deoxy-2-fluoro-glucose, UDP-glucose and UDP. Collectively, these structures provide evidence for the importance of the GlcNAc moiety and its N-acetyl group in donor substrate binding, as well as insight into the role played by the flexible 318-330 loop in substrate binding and product release. In addition, the UDP-CH2-GlcNAc complex reveals a well-defined glycerol molecule poised for nucleophilic attack on the C1 atom of the donor substrate analogue. The position and orientation of this glycerol molecule have allowed us to model the binding of the Manalpha1,3Manbeta1 moiety of the acceptor substrate and, based on the model, to suggest a rationalization for the main determinants of GnT I acceptor specificity. PMID:16769084

  4. The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism.

    PubMed Central

    Gill, J; Rixon, J E; Bolam, D N; McQueen-Mason, S; Simpson, P J; Williamson, M P; Hazlewood, G P; Gilbert, H J

    1999-01-01

    Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10A(CD)) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBD(X)) or fused to the catalytic domain (Pf Xyn10A'), bound to amorphous and bacterial microcrystalline cellulose with a K(a) of 2.5 x 10(5) M(-1). CBD(X) exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A' was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10A(CD); however, Pf Xyn10A' and Pf Xyn10A(CD) exhibited the same activity against soluble substrates. CBD(X) did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10A(CD) when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A', and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10A(CD) via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBD(X) is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase. PMID:10455036

  5. Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2.

    PubMed

    Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin; Kaoud, Tamer S; Arshava, Boris; Dutta, Kaushik; Dalby, Kevin N; Ghose, Ranajeet

    2015-01-01

    The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS. PMID:26054059

  6. Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2

    PubMed Central

    Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin; Kaoud, Tamer S.; Arshava, Boris; Dutta, Kaushik; Dalby, Kevin N.; Ghose, Ranajeet

    2015-01-01

    The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS. PMID:26054059

  7. Photoluminescence study of Be acceptors in GaInNAs epilayers

    NASA Astrophysics Data System (ADS)

    Tsai, Y.; Barman, B.; Scrace, T.; Petrou, A.; Fukuda, M.; Sellers, I. R.; Leroux, M.; Khalfioui, M. A.

    2014-03-01

    We have studied the photoluminescence (PL) spectra from MBE grown GaInNAs epilayers doped p-type with Beryllium acceptors. The measurements were carried out in the 5 K - 70 K temperature range and in magnetic fields (B) up to 7 tesla. The PL spectra contain two features at T = 5 K: The exciton at 1093 meV and a second broader feature at 1058 meV. The intensity of this feature decreases with increasing temperature and disappears completely by 70K while the excitonic feature persists. The emission at 1058meV is identified as the conduction band to Beryllium acceptor transition. If we take into account the binding energy of the exciton [3] we get a value of 23 meV for the Beryllium acceptor binding energy. The acceptor related transition was studied as a function of magnetic field; the energy of this transition has a linear dependence on B with a slope of 055 meV/T. Research supported by Amethyst Research In. through the State of Oklahoma, ONAP program.

  8. The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

    PubMed Central

    Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J.; Sarkar, Mayukh K.; Li, Feng

    2015-01-01

    ABSTRACT Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. IMPORTANCE For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are

  9. Participation of Glutamate-354 of the CP43 Polypeptide in the Ligation of Mn and the Binding of Substrate Water in Photosystem II

    SciTech Connect

    Service, Rachel; Yano, Junko; McConnell, Iain; Hwang, Hong Jin; Niks, Dimitri; Hille, Russ; Wydrzynski, Tom; Burnap, Robert; Hillier, Warwick; Debus, Richard

    2010-09-30

    . The EPR and FTIR data implied that 76 -82 percent of CP43-E354Q PSII centers can achieve the S2 state and that most of these can achieve the S3 state, but no evidence for advancement beyond the S3 state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn4Ca cluster in the S2 state, the FTIR and H218O exchange data show that the mutation strongly influences other properties of the Mn4Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S1 to S2 transition and weakening the binding of both substrate water molecules (or water derived ligands), especially the one that exchanges rapidly in the S3 state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn4Ca cluster in the S1 state as a bridging ligand between two metal ions, but provide no compelling evidence that this residue changes its coordination mode during the S1 to S2 transition. The H218O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S3 state.

  10. Coulomb interaction of acceptors in Cd1-xMnxTe/CdTe quantum dot

    NASA Astrophysics Data System (ADS)

    Kalpana, P.; Reuben, A. Merwyn Jasper D.; Nithiananthi, P.; Jayakumar, K.

    2014-04-01

    The investigation on the effect of confining potential like isotropic harmonic oscillator type potential on the binding and the Coulomb interaction energy of the double acceptors in the presence of magnetic field in a Cd1-xMnxTe/CdTe Spherical Quantum Dot has been made for the Mn ion composition x=0.3 and compared with the results obtained from the square well type potential using variational procedure in the effective mass approximation.

  11. Crystal Structures of An F420-Dependent Glucose-6-Phosphate Dehydrogenase Fgd1 Involved in the Activation of the Anti-Tb Drug Candidate Pa-824 Reveal the Basis of Coenzyme And Substrate Binding

    SciTech Connect

    Bashiri, G.; Squire, C.J.; Moreland, N.J.; Baker, E.N.

    2009-05-11

    The modified flavin coenzyme F{sub 420} is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F{sub 420}-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F{sub 420} for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F{sub 420} and citrate at resolutions of 1.90 and 1.95{angstrom}, respectively. The structure reveals a highly specific F{sub 420} binding mode, which is shared with several other F{sub 420}-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F{sub 420} and is shown to be a competitive inhibitor (IC{sub 50} 43 {micro}m). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F{sub 420} and helps promote a butterfly bend conformation that facilitates F{sub 420} reduction and catalysis.

  12. Vacancy compensation and related donor-acceptor pair recombination in bulk AlN

    SciTech Connect

    Gaddy, BE; Bryan, Z; Bryan, I; Kirste, R; Xie, JQ; Dalmau, R; Moody, B; Kumagai, Y; Nagashima, T; Kubota, Y; Kinoshita, T; Koukitu, A; Sitar, Z; Collazo, R; Irving, DL

    2013-10-14

    A prominent 2.8 eV emission peak is identified in bulk AlN substrates grown by physical vapor transport. This peak is shown to be related to the carbon concentration in the samples. Density functional theory calculations predict that this emission is caused by a donor-acceptor pair (DAP) recombination between substitutional carbon on the nitrogen site and a nitrogen vacancy. Photoluminescence and photoluminescence-excitation spectroscopy are used to confirm the model and indicate the DAP character of the emission. The interaction between defects provides a pathway to creating ultraviolet-transparent AlN substrates for optoelectronics applications. (C) 2013 AIP Publishing LLC.

  13. Molecular insights into the terminal energy acceptor in cyanobacterial phycobilisome.

    PubMed

    Gao, Xiang; Wei, Tian-Di; Zhang, Nan; Xie, Bin-Bin; Su, Hai-Nan; Zhang, Xi-Ying; Chen, Xiu-Lan; Zhou, Bai-Cheng; Wang, Zhi-Xin; Wu, Jia-Wei; Zhang, Yu-Zhong

    2012-09-01

    The linker protein L(CM) (ApcE) is postulated as the major component of the phycobilisome terminal energy acceptor (TEA) transferring excitation energy from the phycobilisome to photosystem II. L(CM) is the only phycobilin-attached linker protein in the cyanobacterial phycobilisome through auto-chromophorylation. However, the underlying mechanism for the auto-chromophorylation of L(CM) and the detailed molecular architecture of TEA is still unclear. Here, we demonstrate that the N-terminal phycobiliprotein-like domain of L(CM) (Pfam00502, LP502) can specifically recognize phycocyanobilin (PCB) by itself. Biochemical assays indicated that PCB binds into the same pocket in LP502 as that in the allophycocyanin α-subunit and that Ser152 and Asp155 play a vital role in LP502 auto-chromophorylation. By carefully conducting computational simulations, we arrived at a rational model of the PCB-LP502 complex structure that was supported by extensive mutational studies. In the PCB-LP502 complex, PCB binds into a deep pocket of LP502 with a distorted conformation, and Ser152 and Asp155 form several hydrogen bonds to PCB fixing the PCB Ring A and Ring D. Finally, based on our results, the dipoles and dipole-dipole interactions in TEA are analysed and a molecular structure for TEA is proposed, which gives new insights into the energy transformation mechanism of cyanobacterial phycobilisome. PMID:22758351

  14. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  15. Coordinated Action of Two Double-Stranded RNA Binding Motifs and an RGG Motif Enables Nuclear Factor 90 To Flexibly Target Different RNA Substrates.

    PubMed

    Schmidt, Tobias; Knick, Paul; Lilie, Hauke; Friedrich, Susann; Golbik, Ralph Peter; Behrens, Sven-Erik

    2016-02-16

    The mechanisms of how RNA binding proteins (RBP) bind to and distinguish different RNA molecules are yet uncertain. Here, we performed a comprehensive analysis of the RNA binding properties of multidomain RBP nuclear factor 90 (NF90) by investigating specifically the functional activities of two double-stranded RNA binding motifs (dsRBM) and an RGG motif in the protein's unstructured C-terminus. By comparison of the RNA binding affinities of several NF90 variants and their modes of binding to a set of defined RNA molecules, the activities of the motifs turned out to be very different. While dsRBM1 contributes little to RNA binding, dsRBM2 is essential for effective binding of double-stranded RNA. The protein's immediate C-terminus, including the RGG motif, is indispensable for interactions of the protein with single-stranded RNA, and the RGG motif decisively contributes to NF90's overall RNA binding properties. Conformational studies, which compared wild-type NF90 with a variant that contains a pseudophosphorylated residue in the RGG motif, suggest that the NF90 C-terminus is involved in conformational changes in the protein after RNA binding, with the RGG motif acting as a central regulatory element. In summary, our data propose a concerted action of all RNA binding motifs within the frame of the full-length protein, which may be controlled by regulation of the activity of the RGG motif, e.g., by phosphorylation. This multidomain interplay enables the RBP NF90 to discriminate RNA features by dynamic and adaptable interactions. PMID:26795062

  16. Engineering of glucoside acceptors for the regioselective synthesis of beta-(1-->3)-disaccharides with glycosynthases.

    PubMed

    Marton, Zsuzanna; Tran, Vinh; Tellier, Charles; Dion, Michel; Drone, Jullien; Rabiller, Claude

    2008-11-24

    Glycosynthase mutants obtained from Thermotogamaritima were able to catalyze the regioselective synthesis of aryl beta-D-Galp-(1-->3)-beta-D-Glcp and aryl beta-D-Glcp-(1-->3)-beta-D-Glcp in high yields (up to 90 %) using aryl beta-D-glucosides as acceptors. The need for an aglyconic aryl group was rationalized by molecular modeling calculations, which have emphasized a high stabilizing interaction of this group by stacking with W312 of the enzyme. Unfortunately, the deprotection of the aromatic group of the disaccharides was not possible without partial hydrolysis of the glycosidic bond. The replacement of aryl groups by benzyl ones could offer the opportunity to deprotect the anomeric position under very mild conditions. Assuming that benzyl acceptors could preserve the stabilizing stacking, benzyl beta-d-glucoside firstly assayed as acceptor resulted in both poor yields and poor regioselectivity. Thus, we decided to undertake molecular modeling calculations in order to design which suitable substituted benzyl acceptors could be used. This study resulted in the choice of 2-biphenylmethyl beta-D-glucopyranoside. This choice was validated experimentally, since the corresponding beta-(1-->3) disaccharide was obtained in good yields and with a high regioselectivity. At the same time, we have shown that phenyl 1-thio-beta-D-glucopyranoside was also an excellent substrate leading to similar results as those obtained with the O-phenyl analogue. The NBS deprotection of the S-phenyl group afforded the corresponding disaccharide quantitatively. PMID:18828996

  17. Alkyl Chlorides as Hydrogen Bond Acceptors

    SciTech Connect

    Nadas, Janos I; Vukovic, Sinisa; Hay, Benjamin

    2012-01-01

    To gain an understanding of the role of an alkyl chloride as a hydrogen bond acceptor, geometries and interaction energies were calculated at the MP2/aug-cc-pVDZ level of theory for complexes between ethyl chloride and representative hydrogen donor groups. The results establish that these donors, which include hydrogen cyanide, methanol, nitrobenzene, pyrrole, acetamide, and N-methylurea, form X-H {hor_ellipsis} Cl hydrogen bonds (X = C, N, O) of weak to moderate strength, with {Delta}E values ranging from -2.8 to -5.3 kcal/mol.

  18. Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences.

    PubMed

    Hebert, Daniel N; Clerico, Eugenia M; Gierasch, Lila M

    2016-09-01

    In this issue of Molecular Cell, Behnke et al. (2016) describe a novel cell-based peptide-binding assay and use it to analyze the binding specificities of the endoplasmic reticulum Hsp70 chaperone and its co-chaperones and to probe their different roles in protein quality control. PMID:27588598

  19. Notch-modifying xylosyltransferase-substrate complexes support an SNi-like retaining mechanism

    PubMed Central

    Yu, Hongjun; Takeuchi, Megumi; LeBarron, Jamie; Kantharia, Joshua; London, Erwin; Bakker, Hans; Haltiwanger, Robert S.; Li, Huilin; Takeuchi, Hideyuki

    2015-01-01

    A major remaining question in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xylosideα1–3 Xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly the Epidermal Growth Factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations where dysregulation of Notch is known to cause cancer or developmental disorders. PMID:26414444

  20. Formation of acceptor centers under the action of redox media on the surface of Cd{sub x}Hg{sub 1-x}Te films

    SciTech Connect

    Varavin, V. S.; Sidorov, G. Yu.

    2009-12-15

    The effect of redox media on the formation of acceptor centers in the Cd{sub x}Hg{sub 1-x}Te films grown by molecular beam epitaxy on the GaAs (301) substrates is studied. When tested for long-term stability, the untreated n-type films do not change their parameters, whereas the treated films exhibit a decrease in the conductivity and the mobility of charge carriers by nearly two orders of magnitude. It is shown that, on the treatments, a source of acceptors is formed at the surface, and the acceptors are most likely mercury vacancies.

  1. H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification

    PubMed Central

    Wu, Haihong; Feigon, Juli

    2007-01-01

    During the biogenesis of eukaryotic ribosomal RNA (rRNA) and spliceosomal small nuclear RNA (snRNA), uridines at specific sites are converted to pseudouridines by H/ACA ribonucleoprotein particles (RNPs). Each H/ACA RNP contains a substrate-specific H/ACA RNA and four common proteins, the pseudouridine synthase Cbf5, Nop10, Gar1, and Nhp2. The H/ACA RNA contains at least one pseudouridylation (ψ) pocket, which is complementary to the sequences flanking the target uridine. In this article, we show structural evidence that the ψ pocket can form the predicted base pairs with substrate RNA in the absence of protein components. We report the solution structure of the complex between an RNA hairpin derived from the 3′ ψ pocket of human U65 H/ACA small nucleolar RNA (snoRNA) and the substrate rRNA. The snoRNA–rRNA substrate complex has a unique structure with two offset parallel pairs of stacked helices and two unusual intermolecular three-way junctions, which together organize the substrate for docking into the active site of Cbf5. The substrate RNA interacts on one face of the snoRNA in the complex, forming a structure that easily could be accommodated in the H/ACA RNP, and explains how successive substrate RNAs could be loaded onto and unloaded from the H/ACA RNA in the RNP. PMID:17412831

  2. The use of N-methylated peptides and depsipeptides to probe the binding of heptapeptide substrates to cAMP-dependent protein kinase.

    PubMed

    Bramson, H N; Thomas, N E; Kaiser, E T

    1985-12-15

    Peptide 1, Leu-Arg-Arg-Ala-Ser-Leu-Gly, is an excellent substrate for cAMP-dependent protein kinase. While the importance of both arginines for effective enzyme-substrate interactions has been shown, it has not been known whether the kinase will catalyze phosphorylation of substrates which contain other than peptide bonds. We report that analogs of peptide 1 which contain depsi linkages replacing selected amide bonds are good protein kinase substrates. Therefore, with the possible exception of the serine amide proton, no peptide 1 amide hydrogens are involved in peptide-peptide or peptide-enzyme hydrogen bonding crucial to defining the high substrate activity of this peptide. It is thus unlikely that peptide 1 is bound by the protein kinase while in an alpha-helical or a beta-turn structure. Three peptides were found to be very poor substrates for protein kinase, those containing N-methyl amino acids in place of Ser5 or Leu6 and a peptide containing Pro in place of Leu6. These peptides are poor substrates for the enzyme possibly because they are unable to adopt a conformation necessary for catalysis of phosphoryl group transfer to occur or due to steric effects in the enzymatic active site. PMID:4066678

  3. tRNA acceptor stem and anticodon bases form independent codes related to protein folding

    PubMed Central

    Carter, Charles W.; Wolfenden, Richard

    2015-01-01

    Aminoacyl-tRNA synthetases recognize tRNA anticodon and 3′ acceptor stem bases. Synthetase Urzymes acylate cognate tRNAs even without anticodon-binding domains, in keeping with the possibility that acceptor stem recognition preceded anticodon recognition. Representing tRNA identity elements with two bits per base, we show that the anticodon encodes the hydrophobicity of each amino acid side-chain as represented by its water-to-cyclohexane distribution coefficient, and this relationship holds true over the entire temperature range of liquid water. The acceptor stem codes preferentially for the surface area or size of each side-chain, as represented by its vapor-to-cyclohexane distribution coefficient. These orthogonal experimental properties are both necessary to account satisfactorily for the exposed surface area of amino acids in folded proteins. Moreover, the acceptor stem codes correctly for β-branched and carboxylic acid side-chains, whereas the anticodon codes for a wider range of such properties, but not for size or β-branching. These and other results suggest that genetic coding of 3D protein structures evolved in distinct stages, based initially on the size of the amino acid and later on its compatibility with globular folding in water. PMID:26034281

  4. Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. [Spinacia oleracea

    SciTech Connect

    Zhu, Genhai; Jensen, R.G. )

    1990-05-01

    The properties of the tight and specific binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO{sub 2} and Mg{sup 2+}, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of ({sup 14}C)CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported. The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg{sup 2+}. Addition of ({sup 14}C)CABP and EDTA stopped binding of Mg{sup 2+} and allowed tight binding of the radiolabel only to sites which were CO{sub 2}/Mg{sup 2+}-activated at that moment. The rate of CO{sub 2} fixation was proportional to the CO{sub 2}/Mg{sup 2+}-activated sites. During light-dependent CO{sub 2} fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg{sup 2+} and CO{sub 2}. Lysis of chloroplasts in media with ({sup 14}C)CABP plus EDTA estimated those carbamylated sites having Mg{sup 2+}. The loss of Rubisco activation during illumination was partially due to the lack of Mg{sup 2+} to stabilize the carbamylated sites.

  5. Time Evolution of the Quaternary Structure of Escherichia Coli Aspartate Transcarbamoylase Upon Reaction With the Natural Substrates And a Slow Tight Binding Inhibitor

    SciTech Connect

    West, J.M.; Xia, J.; Tsuruta, H.; Guo, W.; O'Day, E.M.; Kantrowitz, E.R.

    2009-05-26

    Here, we present a study of the conformational changes of the quaternary structure of Escherichia coli aspartate transcarbamoylase, as monitored by time-resolved small-angle X-ray scattering, upon combining with substrates, substrate analogs, and nucleotide effectors at temperatures between 5 and 22 {sup o}C, obviating the need for ethylene glycol. Time-resolved small-angle X-ray scattering time courses tracking the T {yields} R structural change after mixing with substrates or substrate analogs appeared to be a single phase under some conditions and biphasic under other conditions, which we ascribe to multiple ligation states producing a time course composed of multiple rates. Increasing the concentration of substrates up to a certain point increased the T {yields} R transition rate, with no further increase in rate beyond that point. Most strikingly, after addition of N-phosphonacetyl-l-aspartate to the enzyme, the transition rate was more than 1 order of magnitude slower than with the natural substrates. These results on the homotropic mechanism are consistent with a concerted transition between structural and functional states of either low affinity, low activity or high affinity, high activity for aspartate. Addition of ATP along with the substrates increased the rate of the transition from the T to the R state and also decreased the duration of the R-state steady-state phase. Addition of CTP or the combination of CTP/UTP to the substrates significantly decreased the rate of the T {yields} R transition and caused a shift in the enzyme population towards the T state even at saturating substrate concentrations. These results on the heterotropic mechanism suggest a destabilization of the T state by ATP and a destabilization of the R state by CTP and CTP/UTP, consistent with the T and R state crystallographic structures of aspartate transcarbamoylase in the presence of the heterotropic effectors.

  6. Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

    PubMed

    Ciaccio, P J; Jaiswal, A K; Tew, K D

    1994-06-01

    A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents. PMID:7515059

  7. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin.

    PubMed

    Eudes, Aymerick; Pereira, Jose H; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E K; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D; Loqué, Dominique

    2016-03-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  8. Substrate specificities of mouse heparan sulphate glucosaminyl 6-O-sulphotransferases.

    PubMed

    Smeds, Emanuel; Habuchi, Hiroko; Do, Anh-Tri; Hjertson, Eva; Grundberg, Helena; Kimata, Koji; Lindahl, Ulf; Kusche-Gullberg, Marion

    2003-06-01

    Glycosaminoglycan heparan sulphate interacts with a variety of proteins, such as growth factors, cytokines, enzymes and inhibitors and, thus, influences cellular functions, including adhesion, motility, differentiation and morphogenesis. The interactions generally involve saccharide domains in heparan sulphate chains, with precisely located O-sulphate groups. The 6-O-sulphate groups on glucosamine units, supposed to be involved in various interactions of functional importance, occur in different structural contexts. Three isoforms of the glucosaminyl 6-O-sulphotransferase (6-OST) have been cloned and characterized [H. Habuchi, M. Tanaka, O. Habuchi, K. Yoshida, H. Suzuki, K. Ban and K. Kimata (2000) J. Biol. Chem. 275, 2859-2868]. We have studied the substrate specificities of the recombinant enzymes using various O-desulphated poly- and oligo-saccharides as substrates, and using adenosine 3'-phosphate 5'-phospho[(35)S]sulphate as sulphate donor. All three enzymes catalyse 6-O-sulphation of both -GlcA-GlcNS- and -IdoA-GlcNS- (where GlcA represents D-glucuronic acid, NS the N-sulphate group and IdoA the L-iduronic acid) sequences, with preference for IdoA-containing targets, with or without 2-O-sulphate substituents. 6-OST1 showed relatively higher activity towards target sequences lacking 2-O-sulphate, e.g. the -GlcA-GlcNS- disaccharide unit. Sulphation of such non-O-sulphated acceptor sequences was generally favoured at low acceptor polysaccharide concentrations. Experiments using partially O-desulphated antithrombin-binding oligosaccharide as the acceptor revealed 6-O-sulphation of N-acetylated as well as 3-O-sulphated glucosamine residues with each of the three 6-OSTs. We conclude that the three 6-OSTs have qualitatively similar substrate specificities, with minor differences in target preference. PMID:12611590

  9. Substrate specificities of mouse heparan sulphate glucosaminyl 6-O-sulphotransferases.

    PubMed Central

    Smeds, Emanuel; Habuchi, Hiroko; Do, Anh-Tri; Hjertson, Eva; Grundberg, Helena; Kimata, Koji; Lindahl, Ulf; Kusche-Gullberg, Marion

    2003-01-01

    Glycosaminoglycan heparan sulphate interacts with a variety of proteins, such as growth factors, cytokines, enzymes and inhibitors and, thus, influences cellular functions, including adhesion, motility, differentiation and morphogenesis. The interactions generally involve saccharide domains in heparan sulphate chains, with precisely located O-sulphate groups. The 6-O-sulphate groups on glucosamine units, supposed to be involved in various interactions of functional importance, occur in different structural contexts. Three isoforms of the glucosaminyl 6-O-sulphotransferase (6-OST) have been cloned and characterized [H. Habuchi, M. Tanaka, O. Habuchi, K. Yoshida, H. Suzuki, K. Ban and K. Kimata (2000) J. Biol. Chem. 275, 2859-2868]. We have studied the substrate specificities of the recombinant enzymes using various O-desulphated poly- and oligo-saccharides as substrates, and using adenosine 3'-phosphate 5'-phospho[(35)S]sulphate as sulphate donor. All three enzymes catalyse 6-O-sulphation of both -GlcA-GlcNS- and -IdoA-GlcNS- (where GlcA represents D-glucuronic acid, NS the N-sulphate group and IdoA the L-iduronic acid) sequences, with preference for IdoA-containing targets, with or without 2-O-sulphate substituents. 6-OST1 showed relatively higher activity towards target sequences lacking 2-O-sulphate, e.g. the -GlcA-GlcNS- disaccharide unit. Sulphation of such non-O-sulphated acceptor sequences was generally favoured at low acceptor polysaccharide concentrations. Experiments using partially O-desulphated antithrombin-binding oligosaccharide as the acceptor revealed 6-O-sulphation of N-acetylated as well as 3-O-sulphated glucosamine residues with each of the three 6-OSTs. We conclude that the three 6-OSTs have qualitatively similar substrate specificities, with minor differences in target preference. PMID:12611590

  10. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

    PubMed Central

    Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E.K.; Lee, Taek Soon; Adams, Paul D.; Keasling, Jay D.; Loqué, Dominique

    2016-01-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  11. Donor-acceptor conjugated polymers based on multifused ladder-type arenes for organic solar cells.

    PubMed

    Wu, Jhong-Sian; Cheng, Sheng-Wen; Cheng, Yen-Ju; Hsu, Chain-Shu

    2015-03-01

    Harvesting solar energy from sunlight to generate electricity is considered as one of the most important technologies to address the future sustainability of humans. Polymer solar cells (PSCs) have attracted tremendous interest and attention over the past two decades due to their potential advantage to be fabricated onto large area and light-weight flexible substrates by solution processing at a lower cost. PSCs based on the concept of bulk heterojunction (BHJ) configuration where an active layer comprises a composite of a p-type (donor) and an n-type (acceptor) material represents the most useful strategy to maximize the internal donor-acceptor interfacial area allowing for efficient charge separation. Fullerene derivatives such as [6,6]-phenyl-C61 or 71-butyric acid methyl ester (PCBM) are the ideal n-type materials ubiquitously used for BHJ solar cells. The major effort to develop photoactive materials is numerously focused on the p-type conjugated polymers which are generally synthesized by polymerization of electron-rich donor and electron-deficient acceptor monomers. Compared to the development of electron-deficient comonomers (acceptor segments), the development of electron-rich donor materials is considerably flourishing. Forced planarization by covalently fastening adjacent aromatic and heteroaromatic subunits leads to the formation of ladder-type conjugated structures which are capable of elongating effective conjugation, reducing the optical bandgap, promoting intermolecular π-π interactions and enhancing intrinsic charge mobility. In this review, we will summarize the recent progress on the development of various well-defined new ladder-type conjugated materials. These materials serve as the superb donor monomers to prepare a range of donor-acceptor semi-ladder copolymers with sufficient solution-processability for solar cell applications. PMID:25322211

  12. Acceptor states in heteroepitaxial CdHgTe films grown by molecular-beam epitaxy

    SciTech Connect

    Mynbaev, K. D.; Shilyaev, A. V. Bazhenov, N. L.; Izhnin, A. I.; Izhnin, I. I.; Mikhailov, N. N.; Varavin, V. S.; Dvoretsky, S. A.

    2015-03-15

    The photoluminescence method is used to study acceptor states in CdHgTe heteroepitaxial films (HEFs) grown by molecular-beam epitaxy. A comparison of the photoluminescence spectra of HEFs grown on GaAs substrates (CdHgTe/GaAs) with the spectra of CdHgTe/Si HEFs demonstrates that acceptor states with energy depths of about 18 and 27 meV are specific to CdHgTe/GaAs HEFs. The possible nature of these states and its relation to the HEF synthesis conditions and, in particular, to the vacancy doping occurring under conditions of a mercury deficiency during the course of epitaxy and postgrowth processing are discussed.

  13. Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids

    SciTech Connect

    Haeggblom, M.M.; Rivera, M.D.; Young, L.Y. )

    1993-04-01

    Methanogeneic conditions can promote the biodegradation of a number of halogenated aromatic compounds. This study, using sediments from freshwater and estuarine sites, is an evaluation of the anaerobic biodegradability of monochlorinated phenols and benzoic acids coupled to denitrification, sulfidogenesis, and methanogenesis. The results indicate that chlorinated phenols and benzoic acids are biodegradable under at least one set of anaerobic conditions. Metabolism depends both on the electron acceptor available and on the position of the chlorine substituent. Presence of alternative electron acceptors, nitrate, sulfate, and carbonate, can affect degradation rates and substrate specificities. Since contaminated sites usually have mixtures of wastes, bioremediation efforts may need to consider the activities of diverse anaerobic communities to carry out effective treatment of all components. 37 refs., 4 figs., 4 tabs.

  14. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis.

    PubMed

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T

    2015-01-01

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. PMID:26569229

  15. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis

    PubMed Central

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T.

    2015-01-01

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. PMID:26569229

  16. Purification of Recombinant Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (ACAT1) from H293 Cells and Binding Studies Between the Enzyme and Substrates Using Difference Intrinsic Fluorescence Spectroscopy†

    PubMed Central

    Chang, Catherine CY; Miyazaki, Akira; Dong, Ruhong; Kheirollah, Alireza; Yu, Chunjiang; Geng, Yong; Higgs, Henry N; Chang, Ta-Yuan

    2010-01-01

    Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl CoA binds to ACAT1 with Kd=1.9 μM, and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl CoA elicits a similar spectrum change with much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3beta-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1. PMID:20964445

  17. Genetic evidence for substrate and periplasmic-binding-protein recognition by the MalF and MalG proteins, cytoplasmic membrane components of the Escherichia coli maltose transport system.

    PubMed

    Treptow, N A; Shuman, H A

    1985-08-01

    We isolated mutants of Escherichia coli in which the maltose-binding protein (MBP) is no longer required for growth on maltose as the sole source of carbon and energy. These mutants were selected as Mal+ revertants of a strain which carries a deletion of the MBP structural gene, malE. In one class of these mutants, maltose is transported into the cell independently of MBP by the remaining components of the maltose system. The mutations in these strains map in either malF or malG. These genes code for two of the cytoplasmic membrane components of the maltose transport system. In some of the mutants, MBP actually inhibits maltose transport. We demonstrate that these mutants still transport maltose actively and in a stereospecific manner. These results suggest that the malF and malG mutations result in exposure of a substrate recognition site that is usually available only to substrates bound to MBP. PMID:3894331

  18. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  19. Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity.

    PubMed

    Jemth, Per; Smeds, Emanuel; Do, Anh-Tri; Habuchi, Hiroko; Kimata, Koji; Lindahl, Ulf; Kusche-Gullberg, Marion

    2003-07-01

    Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group. PMID:12702732

  20. Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase.

    PubMed

    Marco-Marín, Clara; Ramón-Maiques, Santiago; Tavárez, Sandra; Rubio, Vicente

    2003-11-28

    We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings

  1. Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase

    PubMed Central

    Patra, Niladri; Ioannidis, Efthymios I.

    2016-01-01

    Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21–22 kcal/mol, in good agreement with experiment (18–19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are

  2. Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase.

    PubMed

    Patra, Niladri; Ioannidis, Efthymios I; Kulik, Heather J

    2016-01-01

    Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21-22 kcal/mol, in good agreement with experiment (18-19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are

  3. A cellulose-binding module of the Trichoderma reesei beta-mannanase Man5A increases the mannan-hydrolysis of complex substrates.

    PubMed

    Hägglund, Per; Eriksson, Torny; Collén, Anna; Nerinckx, Wim; Claeyssens, Marc; Stålbrand, Henrik

    2003-02-27

    Endo-beta-1,4-D-mannanases (beta-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The beta-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5ADeltaCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5ADeltaCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei. PMID:12523968

  4. Aromatic donor-acceptor interactions in non-polar environments.

    PubMed

    Prentice, Giles M; Pascu, Sofia I; Filip, Sorin V; West, Kevin R; Pantoş, G Dan

    2015-05-14

    We have evaluated the strength of aromatic donor-acceptor interactions between dialkyl naphthalenediimide and dialkoxynaphthalene in non-polar environments. (1)H NMR, UV-vis spectroscopy and isothermal titration calorimetry were used to characterise this interaction. We concluded that the strength of donor-acceptor interactions in heptane is sufficient to drive supramolecular assemblies in this and other aliphatic solvents. PMID:25875729

  5. A Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in Formation of the Oxygen-Reactive Complex

    PubMed Central

    Krzyaniak, Matthew D.; Eser, Bekir E.; Ellis, Holly R.; Fitzpatrick, Paul F.; McCracken, John

    2013-01-01

    Tyrosine hydroxylase is a non-heme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin and molecular oxygen. We have used nitric oxide as an O2 surrogate to poise Fe(II) at the catalytic site in an S=3/2, {FeNO}7 form amenable to EPR spectroscopy. 2H-Electron Spin Echo Envelope Modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}7 paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}7 center to the closest deuteron on 6,7-2H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-2H-tyrosine resulted in reorientation of the magnetic axes of the S=3/2 {FeNO}7 center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase. PMID:24168553

  6. Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements.

    PubMed

    Mao, Guanzhong; Chen, Tien-Hao; Srivastava, Abhishek S; Kosek, David; Biswas, Pradip K; Gopalan, Venkat; Kirsebom, Leif A

    2016-01-01

    Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNASer variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1:N+73 base pair for cleavage rate and site selection of pre-tRNASer and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNASer for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNASer, while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection. PMID:27494328

  7. Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements

    PubMed Central

    Srivastava, Abhishek S.; Kosek, David; Biswas, Pradip K.; Gopalan, Venkat; Kirsebom, Leif A.

    2016-01-01

    Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNASer variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1:N+73 base pair for cleavage rate and site selection of pre-tRNASer and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNASer for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNASer, while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection. PMID:27494328

  8. Acceptor impurity activation in III-nitride light emitting diodes

    SciTech Connect

    Römer, Friedhard Witzigmann, Bernd

    2015-01-12

    In this work, the role of the acceptor doping and the acceptor activation and its impact on the internal quantum efficiency (IQE) of a Gallium Nitride (GaN) based multi-quantum well light emitting diode is studied by microscopic simulation. Acceptor impurities in GaN are subject to a high activation energy which depends on the presence of proximate dopant atoms and the electric field. A combined model for the dopant ionization and activation barrier reduction has been developed and implemented in a semiconductor carrier transport simulator. By model calculations, we demonstrate the impact of the acceptor activation mechanisms on the decay of the IQE at high current densities, which is known as the efficiency droop. A major contributor to the droop is the electron leakage which is largely affected by the acceptor doping.

  9. Production of a metal-binding exopolysaccharide by Paenibacillus jamilae using two-phase olive-mill waste as fermentation substrate.

    PubMed

    Morillo, Jose Antonio; Aguilera, Margarita; Ramos-Cormenzana, Alberto; Monteoliva-Sánchez, Mercedes

    2006-09-01

    The present study investigated the use of two-phase olive mill waste (TPOMW) as substrate for the production of exopolysaccharide (EPS) by the endospore-forming bacilli Paenibacillus jamilae. This microorganism was able to grow and produce EPS in aqueous extracts of TPOMW as a unique source of carbon. The effects of substrate concentration and the addition of inorganic nutrients were investigated. Maximal polymer yield in 100-ml batch-culture experiments (2 g l(-1)) was obtained in cultures prepared with an aqueous extract of 20% TPOMW (w/v). An inhibitory effect was observed on growth and EPS production when TPOMW concentration was increased. Nutrient supplementation (nitrate, phosphate, and other inorganic nutrients) did not increase yield. Finally, an adsorption experiment of Pb (II), Cd (II), Cu (II), Zn (II), Co (II), and Ni (II) by EPS is reported. Lead was preferentially complexed by the polymer, with a maximal uptake of 230 mg/g EPS. PMID:16874549

  10. Preparation of oligonucleotides corresponding to the acceptor stem of yeast tRNAPhe and their interaction with yeast ATP(CTP):tRNA nucleotidyltransferase.

    PubMed Central

    Wang, G H; McLaughlin, L W; Sternbach, H; Cramer, F

    1984-01-01

    Seven oligonucleotides corresponding to the 3' and 5' sequences of the acceptor stem of yeast tRNAPhe have been prepared by chemical synthesis, chemical-enzymatic synthesis or by isolation from tRNA hydrolysates. The oligonucleotides have been examined as substrates for phosphodiester bond synthesis in the presence of ATP as catalysed by yeast ATP (CTP): tRNA nucleotidyltransferase. Oligonucleotides which correspond to the sequence of the 3'-strand of the tRNA acceptor stem and possess no secondary structure exhibit little or no activity with the enzyme. The ability of the enzyme to catalyse the synthesis of a phosphodiester linkage using ATP and an oligonucleotide corresponding to the 3'-strand of the acceptor stem is in general dramatically increased when an oligonucleotide corresponding to the sequence of the 5'-strand of tRNA acceptor stem is present. In cases where significant activity was observed kinetic parameters have been determined. Images PMID:6384932

  11. The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site.

    PubMed

    Clifton, Ian J; Ge, Wei; Adlington, Robert M; Baldwin, Jack E; Rutledge, Peter J

    2013-08-19

    Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of β-lactam antibiotics. IPNS converts the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(L-α-aminoadipoyl)-L-cysteinyl-O-methyl-D-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(L-α-aminoadipoyl)-L-cysteinyl-O-methyl-D-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the β-carbon atom of their third residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue. PMID:23860486

  12. Electrochromism of a fused acceptor-donor-acceptor triad covering entire UV-vis and near-infrared regions.

    PubMed

    Yao, Bin; Ye, Xichong; Zhang, Jie; Wan, Xinhua

    2014-10-17

    A novel fused acceptor-donor-acceptor (A-D-A) type panchromatically electrochromic compound was synthesized. It exhibited intensive absorption bands covering entire UV-vis and near-infrared regions upon reduction to the radical anionic state, owing to the simultaneous presence of π*-π* transitions and intervalence charge transfer. PMID:25268224

  13. Acceptor and Excitation Density Dependence of t