Sample records for broad substrate specificity

  1. Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris. (United States)

    Akparov, Valery Kh; Timofeev, Vladimir I; Khaliullin, Ilyas G; Švedas, Vytas; Chestukhina, Galina G; Kuranova, Inna P


    The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs - benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid - were determined by the molecular replacement method at resolutions of 1.57 Å and 1.62 Å to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1' subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1' subsite and the catalytic center.

  2. The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.

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    Ilana Berger

    Full Text Available Cytosolic sulfotransferases (SULTs are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.

  3. Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity* (United States)

    Waz, Shaimaa; Nakamura, Teruya; Hirata, Keisuke; Koga-Ogawa, Yukari; Chirifu, Mami; Arimori, Takao; Tamada, Taro; Ikemizu, Shinji; Nakabeppu, Yusaku; Yamagata, Yuriko


    The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif (i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1. PMID:28035004

  4. Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity. (United States)

    Lovelock, Sarah L; Turner, Nicholas J


    Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids.

  5. Substrate specificity screening of oat (Avena sativa) seeds aminopeptidase demonstrate unusually broad tolerance in S1 pocket. (United States)

    Gajda, Anna D; Pawełczak, Małgorzata; Drag, Marcin


    Aminopeptidases are proteolytic enzymes that remove one amino acid at a time from N-terminus of peptidic substrates. In plants, inhibitors of aminopeptidases can find potential applications in agriculture as herbicides. In this report we have used a library of fluorogenic derivatives of natural and unnatural amino acids for substrate specificity profiling of oat (Avena sativa) aminopeptidase. Interestingly, we have found that this enzyme recognizes effectively among the natural amino acids basic residues like Arg and Lys, hydrophobic Phe, Leu and Met, but also to some extent acidic residues Asp and Glu. In the case of unnatural amino acids hydrophobic residues (hPhe and hCha) and basic hArg were preferentially recognized.

  6. Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

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    Stefano Pascarella


    Full Text Available Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  7. Characterization of an Alkaline Family I.4 Lipase from Bacillus sp. W130-35 Isolated from a Tidal Mud Flat with Broad Substrate Specificity. (United States)

    Kim, Hee Jung; Jung, Won Kyeong; Lee, Hyun Woo; Yoo, Wanki; Kim, T Doohun; Kim, Hoon


    A gene encoding lipolytic enzyme, lip7-3, was isolated from Bacillus sp. W130-35 isolated from a tidal mud flat. The gene encoded a protein of 215 amino acids with a signal peptide composed of 34 amino acid residues. Lip7-3 belonged to the family I.4 lipase and showed its maximal activity at pH 9.0 and 60°C. Its activity increased in the presence of 30% methanol and, remarkably, increased as well to 154.6% in the presence of Ca(2+). Lip7-3 preferred pnitrophenyl octanoate (C8) as a substrate and exhibited broad specificity for short- to longchain fatty acid esters. Additionally, Lip7-3 showed a low degree of enantioselectivity for an S-enantiomer (e.g., (S)-methyl-3-hydroxy-2-methylpropionate). It efficiently hydrolyzed glyceryl tributyrate, but did not hydrolyze glyceryl trioleate, fish oil, or olive oil. Its substrate specificity and activation by the solvent might offer a merit to the biotechnological enzyme applications like transesterification in the production of biodiesel.

  8. LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Deli, Alexandra; Koutsioulis, Dimitrios; Fadouloglou, Vasiliki E.; Spiliotopoulou, Panagiota; Balomenou, Stavroula; Arnaouteli, Sofia; Tzanodaskalaki, Maria; Mavromatis, Konstantinos; Kokkinidis, Michalis; Bouriotis, Vassilis


    The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). Only a few members of this family have been biochemically characterized including N-acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-D-myo-inosityl-2-acetamido-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins), N,N'-diacetylchitobiose (GlcNAc2) and lipoglycopeptide antibiotic de-N-acetylases. All these enzymes share a common feature in that they de-N-acetylate the N-acetyl-D-glucosamine (GlcNAc) moiety of their substrates. The bc1534 gene has previously been cloned and expressed in Escherichia coli. The recombinant enzyme was purified and its 3D structure determined. In this study, the bc3461 gene from B. cereus ATCC14579 was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC 3.5.1.-) and BC3461 were biochemically characterized. The enzymes have different molecular masses, pH and temperature optima and broad substrate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers (GlcNAc2, GlcNAc3 and GlcNAc4), as well as GlcNAc-1P, N-acetyl-d-glucosamine-1 phosphate; GlcNAc-6P, N-acetyl-d-glucosamine-6 phosphate; GalNAc, N-acetyl-d-galactosamine; ManNAc, N-acetyl-d-mannosamine; UDP-GlcNAc, uridine 5'-diphosphate N-acetyl-d-glucosamine. However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B. cereus, N-acetyl-d-glucosaminyl-(β-1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine (GMDP) or N-acetyl-d-GlcN-Nα1-6-d-myo-inositol-1-HPO4-octadecyl (GlcNAc-I-P-C18). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc2 revealed that GlcNAc2 is the favored substrate for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key residues, highlighting

  9. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

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    Marcin Olszewski

    Full Text Available SSB (single-stranded DNA-binding proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein. This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity. The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m is 100.2 °C as shown by differential scanning calorimetry (DSC analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

  10. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity (United States)

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef


    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  11. Characterization of the RokA and HexA broad-substrate-specificity hexokinases from Bacteroides fragilis and their role in hexose and N-acetylglucosamine utilization. (United States)

    Brigham, Christopher J; Malamy, Michael H


    Bacteroides fragilis, a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N-acetyl-D-glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N-acetylmannosamine or N-acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.

  12. Understanding the substrate specificity of conventional calpains. (United States)

    Sorimachi, Hiroyuki; Mamitsuka, Hiroshi; Ono, Yasuko


    Calpains are intracellular Ca(2+)-dependent Cys proteases that play important roles in a wide range of biological phenomena via the limited proteolysis of their substrates. Genetic defects in calpain genes cause lethality and/or functional deficits in many organisms, including humans. Despite their biological importance, the mechanisms underlying the action of calpains, particularly of their substrate specificities, remain largely unknown. Studies show that certain sequence preferences influence calpain substrate recognition, and some properties of amino acids have been related successfully to substrate specificity and to the calpains' 3D structure. The full spectrum of this substrate specificity, however, has not been clarified using standard sequence analysis algorithms, e.g., the position-specific scoring-matrix method. More advanced bioinformatics techniques were used recently to identify the substrate specificities of calpains and to develop a predictor for calpain cleavage sites, demonstrating the potential of combining empirical data acquisition and machine learning. This review discusses the calpains' substrate specificities, introducing the benefits of bioinformatics applications. In conclusion, machine learning has led to the development of useful predictors for calpain cleavage sites, although the accuracy of the predictions still needs improvement. Machine learning has also elucidated information about the properties of calpains' substrate specificities, including a preference for sequences over secondary structures and the existence of a substrate specificity difference between two similar conventional calpains, which has never been indicated biochemically.

  13. Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad-substrate-specificity 2-oxo-acid decarboxylases, in the brewing yeast Saccharomyces pastorianus CBS1483. (United States)

    Bolat, Irina; Romagnoli, Gabriele; Zhu, Feibai; Pronk, Jack T; Daran, Jean-Marc


    The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10, a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10. Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80, which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc-like and one Seub-like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing 'trans' regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.

  14. Designing specificity of protein-substrate interactions

    NARCIS (Netherlands)

    Coluzza, I.; Frenkel, D.


    One of the key properties of biological molecules is that they can bind strongly to certain substrates yet interact only weakly with the very large number of other molecules that they encounter. Using a simple lattice model, we test several methods to design molecule-substrate binding specificity. W

  15. A Novel GH7 Endo-β-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability, Broad Substrate Specificity and Potential Application in the Brewing Industry. (United States)

    Liu, Yun; Dun, Baoqing; Shi, Pengjun; Ma, Rui; Luo, Huiying; Bai, Yingguo; Xie, Xiangming; Yao, Bin


    An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1-20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0-6.0) and excellent stability at pH3.0-8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg-1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.

  16. A Novel GH7 Endo-β-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability, Broad Substrate Specificity and Potential Application in the Brewing Industry.

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    Yun Liu

    Full Text Available An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1-20, a catalytic domain of glycoside hydrolase family 7 (GH7, a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1. The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0-6.0 and excellent stability at pH3.0-8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg-1, moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.

  17. Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases.

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    Bryan M Zhao

    Full Text Available Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P residue, but also the Ser(P and Thr(P residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7, atypical (DUSP3, DUSP14, DUSP22 and DUSP27, viral (variola VH1, and Cdc25 (A-C. Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.

  18. Microbial transglutaminase displays broad acyl-acceptor substrate specificity

    DEFF Research Database (Denmark)

    T. Gundersen, Maria; Keillor, Jeffrey W.; Pelletier, Joelle N.


    The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts...

  19. Signal Peptidase Enzymology and Substrate Specificity Profiling. (United States)

    Dalbey, R E; Pei, D; Ekici, Ö D


    Signal peptidases are membrane proteases that play crucial roles in the protein transport pathway of bacteria. They cleave off the signal peptide from precursor proteins that are membrane inserted by the SecYEG or Tat translocons. Signal peptide cleavage releases the translocated protein from the inner membrane allowing the protein to be exported to the periplasm, outer membrane, or secreted into the medium. Signal peptidases are very important proteins to study. They are unique serine proteases with a Ser-Lys dyad, catalyze cleavage at the membrane surface, and are promising potential antibacterial drug targets. This chapter will focus on the isolation of signal peptidases and the preprotein substrates, as well as describe a peptide library approach for characterizing the substrate specificity.

  20. Identification and specificity of broadly neutralizing antibodies against HIV


    McCoy, Laura E.; Burton, Dennis R.


    Summary Beginning in 2009, studies of the humoral responses of HIV‐positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti‐HIV serum neutralizing responses were crucial in this effort. Equally, developme...

  1. Spatiospectral and picosecond spatiotemporal properties of a broad area operating channeled-substrate-planar laser array (United States)

    Yu, NU; Defreez, Richard K.; Bossert, David J.; Wilson, Geoffrey A.; Elliott, Richard A.


    Spatiospectral and spatiotemporal properties of an eight-element channeled-substrate-planar laser array are investigated in both CW and pulsed operating conditions. The closely spaced CSP array with strong optical coupling between array elements is characterized by a broad area laserlike operation determined by its spatial mode spectra. The spatiotemporal evolution of the near and far field exhibits complex dynamic behavior in the picosecond to nanosecond domain. Operating parameters for the laser device have been experimentally determined. These results provide important information for the evaluation of the dynamic behavior of coherent semiconductor laser arrays.

  2. Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

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    Tyagi Sadhna


    Full Text Available Abstract Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand, although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

  3. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. (United States)

    Guilinger, John P; Pattanayak, Vikram; Reyon, Deepak; Tsai, Shengdar Q; Sander, Jeffry D; Joung, J Keith; Liu, David R


    Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

  4. Crystal Structure and Substrate Specificity of PTPN12

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    Hui Li


    Full Text Available PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12.

  5. Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II. (United States)

    Han, Qian; Cai, Tao; Tagle, Danilo A; Robinson, Howard; Li, Jianyong


    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  6. Substrate Specificity and Structure of Human Aminoadipate Aminotransferase/kynurenine Aminotransferase II

    Energy Technology Data Exchange (ETDEWEB)

    Han,Q.; Cai, T.; Tagle, D.; Robinson, H.; Li, J.


    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to a-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested a-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with a-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  7. Substrate Specificity and Structure of Human aminoadipate aminotransferase/kynurenine aminotransferase II

    Energy Technology Data Exchange (ETDEWEB)

    Han, Q.; Cai, T; Tagle, D; Robinson, H; Li, J


    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  8. Characterizing Protease Specificity: How Many Substrates Do We Need?

    Directory of Open Access Journals (Sweden)

    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  9. Characterizing Protease Specificity: How Many Substrates Do We Need? (United States)

    Schauperl, Michael; Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; Kramer, Christian; Liedl, Klaus R


    Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4') with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  10. [Substrate specificity and kinetic properties of a soluble nucleoside triphosphatase from bovine kidneys]. (United States)

    Sivuk, V F; Rusina, I M; Luchko, T A; Makarchikov, A F


    Soluble nucleoside triphosphatase differing in its properties from all known proteins with NTPase activity was partially purified from bovine kidneys. The enzyme has pH optimum of 7.5, molecular mass of 60 kDa, as estimated by gel chromatography, and shows an absolute dependence on divalent metal ions. NTPase obeyed Michaelis-Menten kinetics in the range of substrate concentration tested from 45 to 440 microM; the apparent Km for inosine-5'-triphosphate was calculated to be 23.3 microM. The enzyme was found to possess a broad substrate specificity, being capable of hydrolyzing various nucleoside-5'-tri- as well as diphosphates.

  11. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

    Directory of Open Access Journals (Sweden)

    Tae-Kyun Oh


    Full Text Available A full-length phytase gene (phy of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR, and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5, an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F, the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

  12. Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities. (United States)

    Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam


    A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

  13. Tissue-specific and substrate-specific mitochondrial bioenergetics in feline cardiac and skeletal muscles

    DEFF Research Database (Denmark)

    Christiansen, Liselotte Bruun; Dela, Flemming; Koch, Jørgen;


    No studies have investigated the mitochondrial function in permeabilized muscle fiber from cats. The aim of this study was to investigate tissue-specific and substrate-specific characteristics of mitochondrial oxidative phosphorylation (OXPHOS) capacity in feline permeabilized oxidative muscle...

  14. Substrate specificity of the neuraminidase of different mumps virus strains. (United States)

    Klamm, H; Pollex, G


    The hydrolysis of neuraminlactose, fetuin, ovomucoid, kappa-caseinglycopeptide and mucine from the bovine submaxillary gland with the neuraminidase (NA) of mumps virus strains Jeryl Lynn, Enders and Berlin 9/76 was investigated to determine the pH-dependence of the reaction with the different substrates. The corresponding curves showed splitting into several peaks with reaction maxima ranging from pH 4.7 to pH 6.7. This occurred probably due to the heterogeneity of the virus samples. The NA of each virus strain had the best reaction affinity to neuraminlactose followed by decreasing affinities to ovomucoid, fetuin and kappa-caseinglycopeptide. The mucine from bovine submaxillary gland was not digested at all. The highest hydrolysis of each substrate was found with the Jeryl Lynn strain, which also possessed the lowest substrate specificity. It was followed by that of strain Enders and finally by isolate Berlin 9/76 which, in turn, had the highest substrate specificity. The lower substrate specificity of mumps virus NA seemed to correlate with a lower degree of its cytopathogenicity for hamster ependyma cells.

  15. Molecular Cloning and Characterization of a Broad Substrate Terpenoid Oxidoreductase from Artemisia annua

    NARCIS (Netherlands)

    Ryden, Anna-Margareta; Ruyter-Spira, Carolien; Litjens, Ralph; Takahashi, Shunji; Quax, Wim; Osada, Hiroyuki; Bouwmeester, Harro; Kayser, Oliver


    From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids, a

  16. Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.

    NARCIS (Netherlands)

    Ryden, A.M.; Ruyter-Spira, C.P.; Litjens, R.; Takahashi, S.; Quax, W.J.; Osada, H.; Bouwmeester, H.J.; Kayser, O.


    From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids,

  17. Substrate specificity within a family of outer membrane carboxylate channels.

    Directory of Open Access Journals (Sweden)

    Elif Eren


    Full Text Available Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  18. Surprisingly high substrate specificities observed in complex biofilms

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Kindaichi, Tomonori; Kragelund, Caroline;

    The behavior of microorganisms in natural ecosystems (e.g. biofilms) differs significantly from laboratory studies. In nature microorganisms experience alternating periods of surplus nutrients, nutrient-limitation, and starvation. Literature data suggests that to survive and compete successfully......, microorganisms can regulate their metabolism expressing wide range of uptake and catabolic systems. However, ecophysiological studies of natural biofilms indicate that bacteria are very specialized in their choice of substrate, so even minor changes in substrate composition can affect the community composition...... by selection for different specialized species. We hypothesized that bacteria growing in natural environment express strongly conserved substrate specificity which is independent on short-term (few hours) variations in growth conditions. In this study, biofilm from Aalborg wastewater treatment plant was used...

  19. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C. (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin


    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  20. A loop of coagulation factor VIIa influencing macromolecular substrate specificity

    DEFF Research Database (Denmark)

    Bjelke, Jais R; Persson, Egon; Rasmussen, Hanne B;


    Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa....../Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation...

  1. Relative Specificity: All Substrates Are Not Created Equal

    Directory of Open Access Journals (Sweden)

    Yan Zeng


    Full Text Available A biological molecule, e.g., an enzyme, tends to interact with its many cognate substrates, targets, or partners differentially. Such a property is termed relative specificity and has been proposed to regulate important physiological functions, even though it has not been examined explicitly in most complex biochemical systems. This essay reviews several recent large-scale studies that investigate protein folding, signal transduction, RNA binding, translation and transcription in the context of relative specificity. These results and others support a pervasive role of relative specificity in diverse biological processes. It is becoming clear that relative specificity contributes fundamentally to the diversity and complexity of biological systems, which has significant implications in disease processes as well.

  2. Mechanisms of Yersinia YopO kinase substrate specificity (United States)

    Lee, Wei Lin; Singaravelu, Pavithra; Wee, Sheena; Xue, Bo; Ang, Khay Chun; Gunaratne, Jayantha; Grimes, Jonathan M.; Swaminathan, Kunchithapadam; Robinson, Robert C.


    Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO’s substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis. PMID:28051168

  3. Molecular mechanism of strict substrate specificity of an extradiol dioxygenase, DesB, derived from Sphingobium sp. SYK-6.

    Directory of Open Access Journals (Sweden)

    Keisuke Sugimoto

    Full Text Available DesB, which is derived from Sphingobium sp. SYK-6, is a type II extradiol dioxygenase that catalyzes a ring opening reaction of gallate. While typical extradiol dioxygenases show broad substrate specificity, DesB has strict substrate specificity for gallate. The substrate specificity of DesB seems to be required for the efficient growth of S. sp. SYK-6 using lignin-derived aromatic compounds. Since direct coordination of hydroxyl groups of the substrate to the non-heme iron in the active site is a critical step for the catalytic reaction of the extradiol dioxygenases, the mechanism of the substrate recognition and coordination of DesB was analyzed by biochemical and crystallographic methods. Our study demonstrated that the direct coordination between the non-heme iron and hydroxyl groups of the substrate requires a large shift of the Fe (II ion in the active site. Mutational analysis revealed that His124 and His192 in the active site are essential to the catalytic reaction of DesB. His124, which interacts with OH (4 of the bound gallate, seems to contribute to proper positioning of the substrate in the active site. His192, which is located close to OH (3 of the gallate, is likely to serve as the catalytic base. Glu377' interacts with OH (5 of the gallate and seems to play a critical role in the substrate specificity. Our biochemical and structural study showed the substrate recognition and catalytic mechanisms of DesB.

  4. Determination of substrate specificity of polyamine transporters in roseobacter species (United States)

    Madhuri, S.; Mou, X.


    Polyamines, such as cadaverine, putrescine, spermidine, spermine and norspermine are a class of dissolved organic nitrogen (DON) that is ubiquitously found in marine environments. Intracellular polyamines are important in a variety of biological reactions, such as nucleic acid synthesis and protein synthesis. Free polyamines in seawater can be transported into bacterial cells by ABC transporter systems, each of which consists of four components including one substrate binding protein, one ATPase and two permeases. In silico analysis of marine bacterial genomes has revealed that roseobacter, a numerically and ecologically important taxa of marine bacteria, have at least two sets of polyamine transporter genes. This study was to examine the potential preference of roseobacter to different polyamine compounds and the substrate specificity of different polyamine transporters. Eleven roseobacter species, which genomes have been sequenced, were grown in defined media supplied with single polyamine compound as the sole carbon and nitrogen source. Growth assay showed a small number of roseobacter isolates to be generalist showing no preference among the tested polyamines (Ruegeria pomeroyi DSS-3, Roseovarius sp. TM1035, Roseovarius nubinhibens ISM, Jannaschia sp. CCS1 and Sagittula stellata E-37), whereas other isolates were specilists and were specific on polyamine compounds (Roseobacter sp. CCS2 and Roseobacter denitrificans OCh 114). Primers that probe poly-1 and pot-D genes, the two genes that encode common polyamine-binding genes of polyamine transporter systems were designed using net primer and primer design program. The specificity of the primers was validated by PCR followed by amplicon sequencing. Single step reverse transcription quantitative polymerase chain reactions (RT-qPCR) was performed to investigate substrate specificity of poly-1 and pot-D genes. Key-words Roseobacter, polyamine, polyamine transporter, dissolved organic nitrogen

  5. Structural determinants of tobacco vein mottling virus protease substrate specificity. (United States)

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S


    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  6. Directed evolution of the substrate specificity of biotin ligase. (United States)

    Lu, Wei-Cheng; Levy, Matthew; Kincaid, Rodney; Ellington, Andrew D


    We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA6-40 and BirA6-47 , showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA6-40 and the single substitution variant BirAM157T revealed that they had twoto threefold higher kcat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications.

  7. Substrate specificity of glucokinase and fructokinase of several conifer species

    Directory of Open Access Journals (Sweden)

    Fritz Bergmanni


    Full Text Available Annual plant species were found to have several distinct classes of hexokinases which are specific for different hexoses, such as glucose, fructose and mannose. In conifers one isozyme of hexokinase could be found in genetic studies if only glucose was employed as substrate. If fructose was substituted for glucose, another isozyme zone different from the common hexokinase could be observed in zymograms of extracts from seed tissues of Norway spruce, Scots pine and silver fir. Hence these three conifer species possess at least two different hexokinases, glucokinase and fructokinase.

  8. African Cultural and Literary Specificity in the Broad Translation Quality Debate

    Directory of Open Access Journals (Sweden)

    Wanchia T. Neba


    Full Text Available Beyond revisiting the byzantine and seemingly inconclusive debate on translation quality assurance and assessment, this article investigates the extent of an across-the-board applicability of existing quality assessment frameworks to the broad translation quality debate, against a strong backdrop of culture-specificity. It, first and foremost, exemplifies cultural and literary specificity through linguistically open-ended African creative writing, examines the variegated concept of translation, the volatile concept of translation quality assurance and assessment, outlines constraints to the assurance and assessment of this translation quality, and importantly portrays the preponderant place of metrics, rubrics and models in quality assurance and assessment. Secondly and finally, using a blend of literary and translation theories and strategies, it then qualitatively demonstrates from existing evidence, that quality assurance with its acquiesced formulae will continue to be at the mercy of incontestable contextualised cultural specificity – being of necessity a ‘provincialised’ and ‘balkanised’ activity.

  9. Molecular Determinants of Substrate Specificity in Plant 5-Methylthioadenosine Nucleosidases

    Energy Technology Data Exchange (ETDEWEB)

    Siu,K.; Lee, J.; Sufrin, J.; Moffatt, B.; McMillan, M.; Cornell, K.; Isom, C.; Howell, L.


    5?-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5?-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5?-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Angstroms resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5?-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5?-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pKa of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and

  10. Substrate specificity of allelic variants of the TAP peptide transporter. (United States)

    Heemels, M T; Ploegh, H L


    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant.

  11. Detailed characterization of the substrate specificity of mouse wax synthase. (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni


    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  12. Substrate specificity of Rhbg: ammonium and methyl ammonium transport. (United States)

    Nakhoul, Nazih L; Abdulnour-Nakhoul, Solange M; Boulpaep, Emile L; Rabon, Edd; Schmidt, Eric; Hamm, L Lee


    Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg.

  13. Redesigning the substrate specificity of an enzyme: isocitrate dehydrogenase. (United States)

    Doyle, S A; Fung, S Y; Koshland, D E


    Despite the structural similarities between isocitrate and isopropylmalate, isocitrate dehydrogenase (IDH) exhibits a strong preference for its natural substrate. Using a combination of rational and random mutagenesis, we have engineered IDH to use isopropylmalate as a substrate. Rationally designed mutations were based on comparison of IDH to a similar enzyme, isopropylmalate dehydrogenase (IPMDH). A chimeric enzyme that replaced an active site loop-helix motif with IPMDH sequences exhibited no activity toward isopropylmalate, and site-directed mutants that replaced IDH residues with their IPMDH equivalents only showed small improvements in k(cat). Random mutants targeted the IDH active site at positions 113 (substituted with glutamate), 115, and 116 (both randomized) and were screened for activity toward isopropylmalate. Six mutants were identified that exhibited up to an 8-fold improvement in k(cat) and increased the apparent binding affinity by as much as a factor of 80. In addition to the S113E mutation, five other mutants contained substitutions at positions 115 and/or 116. Most small hydrophobic substitutions at position 116 improved activity, possibly by generating space to accommodate the isopropyl group of isopropylmalate; however, substitution with serine yielded the most improvement in k(cat). Only two substitutions were identified at position 115, which suggests a more specific role for the wild-type asparagine residue in the utilization of isopropylmalate. Since interactions between neighboring residues in this region greatly influenced the effects of each other in unexpected ways, structural solutions were best identified in combinations, as allowed by random mutagenesis.

  14. Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    LIU Xian-jin; YAN Chun-rong; LIU Yuan; YU Xiang-yang; ZHANG Cun-zheng


    Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL-1 and an I50 value of 0.182 ug mL-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 ug mL-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semi-quantitation determination of the above former five organophosphorus pesticides.

  15. Specificity of Mucor miehei lipase on methyl ester substrates

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    Pina, M.


    Full Text Available Fatty acid methyl esters constitute a good substrate for the characterization of lipase typospecificity. In the present work, the hydrolytic action of lipase from Mucor miehei was studied. It was demonstrated that this lipase preferentially catalyses the hydrolysis of fatty acid methyl esters with small number of double bonds. It was also found that this lipase shows a specificity in the hydrolysis of fatty acid methyl esters with short aliphatic chain.Los esteres metílicos de ácidos grasos constituyen un buen sustrato para la caracterización de tipos de especificidad de lipasa. En el presente trabajo, se estudió la acción hidrolítica de lipasa de Mucor miehei. Se demostró que esta lipasa cataliza preferencialmente la hidrólisis de esteres metílicos de ácidos grasos con número pequeño de dobles enlaces. Se encontró también que esta lipasa muestra una especificidad en la hidrólisis de ásteres metílicos de ácidos grasos con cadena alifática corta.

  16. Substrate specificity in enzymatic fluorination. The fluorinase from Streptomyces cattleya accepts 2'-deoxyadenosine substrates. (United States)

    Cobb, Steven L; Deng, Hai; McEwan, Andrew R; Naismith, James H; O'Hagan, David; Robinson, David A


    The fluorinase enzyme from Streptomyces cattleya displays an unusual ability in biocatalysis in that it forms a C-F bond. We now report that the enzyme will accept 2'-deoxyadenosine in place of adenosine substrates, and structural evidence reveals a reorganisation in hydrogen bonding to accommodate this substrate series. It emerges from this study that the enzyme does not require a planar ribose conformation of the substrate to catalyse C-F bond formation.

  17. Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1. (United States)

    Dewitte, Griet; Walmagh, Maarten; Diricks, Margo; Lepak, Alexander; Gutmann, Alexander; Nidetzky, Bernd; Desmet, Tom


    UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource.

  18. Broad-specificity immunoassays for sulfonamide detection: immunochemical strategy for generic antibodies and competitors. (United States)

    Franek, Milan; Diblikova, Iva; Cernoch, Ivo; Vass, Maria; Hruska, Karel


    Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.

  19. Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

    NARCIS (Netherlands)

    Kamerbeek, NM; Olsthoorn, AJJ; Fraaije, MW; Janssen, DB; Kamerbeek, Nanne M.; Olsthoorn, Arjen J.J.


    The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of ca

  20. Substrate specificity of papain dynamic structures for peptides consisting of 8-10 GLY residues (United States)

    Nishiyama, Katsuhiko


    We investigated the substrate specificity of papain dynamic structures for peptides of 8-10 glycine residues (8-10GLY) via molecular dynamics and docking simulations. The substrate specificity of papain for 8-10GLY fluctuated considerably with time. There were several residues that were different among those that had a significant impact on binding (RESIDUES_IMPACT) with 10GLY, 9GLY, and 8GLY. Modification of these different residues should allow for control of substrate specificity, providing a framework for modifying substrate specificity in papain and other enzymes.

  1. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+). (United States)

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J


    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.

  2. Switching substrate specificity of AMT/MEP/ Rh proteins (United States)

    Neuhäuser, Benjamin; Dynowski, Marek; Ludewig, Uwe


    In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH4+, NH3 + H+, NH4+ + H+ or NH3, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH3 transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH3 across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH3 transport through the central subunit pore is suggested. PMID:25483282

  3. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

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    Caroline Lund Dahlberg

    Full Text Available BACKGROUND: Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. CONCLUSIONS/SIGNIFICANCE: The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  4. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family

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    Chaikuad Apirat


    Full Text Available Abstract Backround Aspartyl aminopeptidase (DNPEP, with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

  5. Characterization of the Kluyveromyces marxianus strain DMB1 YGL157w gene product as a broad specificity NADPH-dependent aldehyde reductase. (United States)

    Akita, Hironaga; Watanabe, Masahiro; Suzuki, Toshihiro; Nakashima, Nobutaka; Hoshino, Tamotsu


    The open reading frame YGL157w in the genome of the yeast Kluyveromyces marxianus strain DMB1 encodes a putative uncharacterized oxidoreductase. However, this protein shows 46% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase, which exhibits broad substrate specificity for aldehydes. In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (branched-chain and linear) and aromatic aldehydes. The optimal pH for methylglyoxal reduction was 5.5. With methylglyoxal as a substrate, the optimal temperature for enzyme activity at pH 5.5 was 45°C. The enzyme retained more than 70% of its activity after incubation for 30 min at temperatures below 35°C or at pHs between 5.5 and 9.0. In addition, the KmGRE2-overexpressing E. coli showed improved growth when cultivated in cedar hydrolysate, as compared to cells not expressing the enzyme. Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells.

  6. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.


    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  7. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

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    Woo-Suk Jung

    Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  8. Structural Evidence of Substrate Specificity in Mammalian Peroxidases (United States)

    Sheikh, Ishfaq Ahmed; Singh, Amit Kumar; Singh, Nagendra; Sinha, Mau; Singh, S. Baskar; Bhushan, Asha; Kaur, Punit; Srinivasan, Alagiri; Sharma, Sujata; Singh, Tej P.


    The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN–) has been determined at 2.4Å resolution. It revealed that the SCN– ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN– ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN– forms a hydrogen bond with a water (Wat) molecule at position 6′. This water molecule is stabilized by two hydrogen bonds with Gln423 Nε2 and Phe422 oxygen. In contrast, the placement of the SCN– ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln423, Phe422 oxygen, and Wat6′ in LPO is occupied primarily by the side chain of Phe407 in MPO due to an entirely different conformation of the loop corresponding to the segment Arg418–Phe431 of LPO. This arrangement in MPO does not favor a similar orientation of the SCN– ion. The orientation of the catalytic product OSCN– as reported in the structure of LPO·OSCN– is similar to the orientation of SCN– in the structure of LPO·SCN–. Similarly, in the structure of LPO·SCN–·CN–, in which CN– binds at Wat1, the position and orientation of the SCN– ion are also identical to that observed in the structure of LPO·SCN. PMID:19339248

  9. Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1. (United States)

    Majiduddin, Fahd K; Palzkill, Timothy


    Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by beta-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 beta-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A beta-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of beta-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

  10. Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

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    Willis, Mark A.; Zhuang, Zhihao; Song, Feng; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat (UNM); (IIT); (UMBI)


    The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.

  11. Engineering Substrate Preference in Subtilisin: Structural and Kinetic Analysis of a Specificity Mutant

    Energy Technology Data Exchange (ETDEWEB)

    Ruan, Biao; London, Viktoriya; Fisher, Kathryn E.; Gallagher, D. Travis; Bryan, Philip N. (UMBI)


    Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k{sub cat} for the reaction. This also leads to strong product inhibition of substrate

  12. Long-term nonprogression and broad HIV-1-specific proliferative T-cell responses

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    Nesrina eImami


    Full Text Available Complex mechanisms underlying the maintenance of fully functional, proliferative, HIV-1-specific T-cell responses involve processes from early T-cell development through to the final stages of T-cell differentiation and antigen recognition. Virus-specific proliferative CD4 and CD8 T-cell responses, important for the control of infection, are observed in some HIV-1+ patients during early stages of disease, and are maintained in long-term nonprogressing subjects. In the vast majority of HIV-1+ patients, full immune functionality is lost when proliferative HIV-1-specific T-cell responses undergo a variable progressive decline throughout the course of chronic infection. This appears irreparable despite administration of potent combination antiretroviral therapy, which to date is non-curative, necessitating life-long administration and the development of effective, novel, therapeutic interventions. While a sterilising cure, involving clearance of virus from the host, remains a primary aim, a functional cure may be a more feasible goal with considerable impact on worldwide HIV-1 infection. Such an approach would enable long-term co-existence of host and virus in the absence of toxic and costly drugs. Effective immune homeostasis coupled with a balanced response appropriately targeting conserved viral antigens, in a manner that avoids hyperactivation and exhaustion, may prove to be the strongest correlate of durable viral control. This review describes novel concepts underlying full immune functionality in the context of HIV-1 infection, which may be utilised in future strategies designed to improve upon existing therapy. The aim will be to induce long-term nonprogressor or elite controller status in every infected host, through immune-mediated control of viraemia and reduction of viral reservoirs, leading to lower HIV-1 transmission rates.

  13. Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

    Directory of Open Access Journals (Sweden)

    Nitish K Mishra

    Full Text Available BACKGROUND: Membrane transport proteins (transporters move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task. RESULTS: Support vector machine (SVM-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM, were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC of 0.49 and a receiver operating characteristic area under the curve (AUC of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset. CONCLUSIONS: Our analyses suggest that evolutionary information (i.e., the PSSM and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models

  14. YqhD. A broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Jarboe, Laura R. [Iowa State Univ., Ames, IA (United States). Dept. of Chemical and Biological Engineering


    The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor. (orig.)

  15. Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates. (United States)

    Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F


    The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

  16. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase (United States)

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian


    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  17. Tissue-specific and substrate-specific mitochondrial bioenergetics in feline cardiac and skeletal muscles

    DEFF Research Database (Denmark)

    Christiansen, Liselotte Bruun; Dela, Flemming; Koch, Jørgen;


    fibers. Biopsies of left ventricular cardiac muscle and soleus muscle, a type I-rich oxidative skeletal muscle, were obtained from 15 healthy domestic cats. Enzymatic activity of citrate synthase (CS), a biomarker of mitochondrial content, was measured. Mitochondrial OXPHOS capacity with various kinds...... of non-fatty-acid substrates and fatty-acid substrate in permeabilized muscle fiber was measured by using high-resolution respirometry. CS activity in the heart was 3 times higher than in the soleus muscle. Mitochondrial state 3 respiration, ADP-stimulated respiration, with complex I-linked and complex I...

  18. Global identification and analysis of isozyme-specific possible substrates crosslinked by transglutaminases using substrate peptides in mouse liver fibrosis (United States)

    Tatsukawa, Hideki; Tani, Yuji; Otsu, Risa; Nakagawa, Haruka; Hitomi, Kiyotaka


    The transglutaminase (TG) family comprises eight isozymes that form the isopeptide bonds between glutamine and lysine residues and contribute to the fibrotic diseases via crosslinking-mediated stabilization of ECM and the activation of TGF-β in several tissues. However, despite a growing body of evidence implicating TG2 as a key enzyme in fibrosis, the causative role of TG2 and the involvement of the other isozymes have not yet been fully elucidated. Therefore, here we clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific possible substrates for both TG1 and TG2 using their substrate peptides in mouse fibrotic liver. We found that TG1 activity was markedly enhanced intracellularly over a widespread area, whereas TG2 activity increased in the extracellular space. In total, 43 and 42 possible substrates were identified for TG1 and TG2, respectively, as involved in chromatin organization and cellular component morphogenesis. These included keratin 18, a biomarker for hepatic injury, which was accumulated in the fibrotic liver and showed the partly similar distribution with TG1 activity. These findings suggest that TG1 activity may be involved in the functional modification of intracellular proteins, whereas TG2 activity contributes to the stabilization of extracellular proteins during liver fibrosis. PMID:28327670

  19. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

    DEFF Research Database (Denmark)

    Larsen, Katrine S; Østergaard, Henrik; Bjelke, Jais R;


    of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather...... promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen...... for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered...

  20. A mesoscopic stochastic model for the specific consumption rate in substrate-limited microbial growth (United States)


    The specific consumption rate of substrate, as well as the associated specific growth rate, is an essential parameter in the mathematical description of substrate-limited microbial growth. In this paper we develop a completely new kinetic model of substrate transport, based on recent knowledge on the structural biology of transport proteins, which correctly describes very accurate experimental results at near-zero substrate concentration values found in the literature, where the widespread Michaelis-Menten model fails. Additionally, our model converges asymptotically to Michaelis-Menten predictions as substrate concentration increases. Instead of the single active site enzymatic reaction of Michaelis-Menten type, the proposed model assumes a multi-site kinetics, simplified as an apparent all-or-none mechanism for the transport, which is controlled by means of the local substrate concentration in the close vicinity of the transport protein. Besides, the model also assumes that this local concentration is not equal to the mean substrate concentration experimentally determined in the culture medium. Instead, we propose that it fluctuates with a mostly exponential distribution of Weibull type. PMID:28187189

  1. A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine. (United States)

    Ibarrola, Nieves; Molina, Henrik; Iwahori, Akiko; Pandey, Akhilesh


    Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.

  2. Study on Substrate Specificity at Subsites for Severe Acute Respiratory Syndrome Coronavirus 3CL Protease

    Institute of Scientific and Technical Information of China (English)

    Yu-Fei SHAN; Gen-Jun XU


    Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2'. The deletion of either P4 or P2' residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S 1' subsite, we have found that bulky residues such as Tyr and Trp were also acceptable.In addition, based on both peptide-based assay and autocleavage assay, Ile at the P1' position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S 1' subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2' position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.

  3. Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site. (United States)

    Kühn-Wache, Kerstin; Bär, Joachim W; Hoffmann, Torsten; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich


    Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P₁-position for proline, alanine, glycine and serine with 5.0 x 10⁵ M⁻¹ s⁻¹, 1.8 x 10⁴ M⁻¹ s⁻¹, 3.6 x 10² M⁻¹ s⁻¹, 1.1 x 10² M⁻¹ s⁻¹, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P₁-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P₁-position with 4 x 10⁵ M⁻¹ s⁻¹, 9.5 x 10⁵ M⁻¹ s⁻¹ and 2.1 x 10⁵ M⁻¹ s⁻¹, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P₄'-P₈'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

  4. The specificity of viral sialidases. The use of oligosaccharide substrates to probe enzymic characteristics and strain-specific differences. (United States)

    Corfield, A P; Wember, M; Schauer, R; Rott, R


    1. The action of sialidases from Newcastle disease virus (NDV), influenza A2 virus (IA2V) and fowl plague virus (FPV) on sialyloligosaccharide substrates containing alpha 2-3, alpha 2-6 or alpha 2-8 linkages was studied. 2. In all cases 2-3-linked sialic acids were preferentially released. Compared with II6Neu5AcLac, all 2-6-linked substrates, including sialyl-N-acetyllactosamine and its asparaginyl derivative, a urinary hexasaccharide and Neu5Ac(2-6)GalNAc were cleaved at improved rates by NDV and less by FPV sialidases. In the case of IA2V sialidase the asparaginyl oligosaccharide was very poorly cleaved, illustrating a variation in viral strain specificity. 3. A decrease in relative rates was observed in the order NDV greater than IA2V greater than FPV for substrates with 2-3 linkages relative to II6Neu5AcLac. The greatest relative rate was 470-fold higher. The 2-3-linked sialyl-N-acetyllactosaminylasparagine and IV3Neu5AcLcOse4 were poor substrates for the IA2V sialidase, but the rates were greater than with the 2-6 linked substrates. 4. The ganglioside substrate II3Neu5AcLacCer showed lower activity than its oligosaccharide analogue, but neither II3Neu5AcGgOse4Cer nor its oligosaccharide were substrates. 5. The Km values for 2-6-linked substrates were generally of the order 10 mM while those for the 2-3-linked substrates were approximately 1 mM. The V values were consistently higher for the 2-3-linked substrates. IV3Neu5AcLcOse4 showed high Km and very high V values, while the 2-8-linked disialyllactose showed this trend only with NDV enzyme, the IA2V and FPV sialidases exhibiting high Km and low V values. 6. The results are discussed in the light of the current knowledge of viral sialidase specificity and relative to the binding of virus particles to cell surfaces.

  5. Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

    Directory of Open Access Journals (Sweden)

    Huimin Liu


    Full Text Available Cysteine protease 1 precursor from Zea mays (zmCP1 is classified as a member of the C1A family of peptidases (papain-like cysteine protease in MEROPS (the Peptidase Database. The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe. Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.

  6. Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases. (United States)

    Kochinyan, Samvel; Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Xu, Jie; Xu, Ming-Qun


    Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.

  7. [Substrate specificity of carotenoid 3',4'-desaturase from Deinococcus radiodurans]. (United States)

    Sun, Zongtao; Tian, Bing; Shen, Shaochuan; Hu, Yuejin


    To examine the substrate specificity of carotenoid 3',4'-desaturase (DR2250) from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBI(Eu) and (or) pRK-CRTC and (or) pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 (1').

  8. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice. (United States)

    McGuire, Andrew T; Gray, Matthew D; Dosenovic, Pia; Gitlin, Alexander D; Freund, Natalia T; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B; Glenn, Jolene; Seaman, Michael S; Schief, William R; Strong, Roland K; Nussenzweig, Michel C; Stamatatos, Leonidas


    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

  9. HIV-1 clade C escapes broadly neutralizing autologous antibodies with N332 glycan specificity by distinct mechanisms. (United States)

    Deshpande, Suprit; Patil, Shilpa; Kumar, Rajesh; Hermanus, Tandile; Murugavel, Kailapuri G; Srikrishnan, Aylur K; Solomon, Suniti; Morris, Lynn; Bhattacharya, Jayanta


    The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms.

  10. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.


    Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  11. Site specific isolated nanostructure array formation on a large area by broad ion beam without any mask and resist

    Energy Technology Data Exchange (ETDEWEB)

    Karmakar, Prasanta, E-mail: [Variable Energy Cyclotron Centre, 1/AF, Bidhannagar, Kolkata 700064 (India); Satpati, Biswarup [Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064 (India)


    We report the formation of isolated nanostructure arrays on a large area via broad ion beam implantation without the aid of any mask or resist. Desired ions have been implanted at specific locations of the prefabricated silicon ripple or triangular structures by exploiting the variation of local ion impact angles. We have shown that the implantation of Fe ions on an O{sup +} ions induced pre fabricated triangular shaped patterned Si surface results in a self-organized periodic array of striped magnetic nanostructures having several micron length and about 50 nm width arranged with a spacial separation of ∼200 nm. The morphology, composition, crystalline structure, and magnetic property of these nanopatterns have been analyzed using high-resolution cross-sectional transmission electron microscopy and atomic force microscopy. A geometrical model has been proposed to explain the fundamental features of such ion-induced nanopattern structures.

  12. Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray (United States)

    Cornett, E.M.; Dickson, B.M.; Vaughan, R.M.; Krishnan, S.; Trievel, R.C.; Strahl, B.D.; Rothbart, S.B.


    The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the “histone code” hypothesis, we reveal a strong influenceof adjacent and,surprisingly,distant histonePTMs onthe ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes. PMID:27423856

  13. Structural view and substrate specificity of papain-like protease from avian infectious bronchitis virus. (United States)

    Kong, Lingying; Shaw, Neil; Yan, Lingming; Lou, Zhiyong; Rao, Zihe


    Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys(48)- and Lys(63)-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys(101), His(264), and Asp(275) is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the β-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus.

  14. Most substrates suitable if you adapt the watering and fertiliser : take note of specific plants needs

    NARCIS (Netherlands)

    Heuvelink, E.; Kierkels, T.


    The diversity in substrates keeps growing. You can't say that one is better than the other if you take into account their characteristics and adapt the watering and fertilisation. But you also need take into account the specific requirements of the plant which we'll discuss in this article.

  15. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis. (United States)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram; Madsen, Thomas Daugbjerg; Gerken, Thomas A; Vester-Christensen, Malene B; Wandall, Hans H; Bennett, Eric Paul; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik


    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

  16. Development of a monoclonal antibody-based broad-specificity ELISA for fluroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds (United States)

    Development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR) and ofloxacin (OFL) were conjugated to...

  17. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anke K Trilling

    Full Text Available BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA tests and soluble antigen by surface plasmon resonance (SPR analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp. The highest affinity VHH had a dissociation constant (KD of 4 × 10(-10 M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

  18. Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance. (United States)

    Kim, Jun-Hwan; Lee, Kang-Keun; Sun, Younguk; Seo, Gang-Jin; Cho, Sung Suk; Kwon, Suk Hyung; Kwon, Suk-Tae


    The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

  19. Understanding the substrate specificity of the heparan sulfate sulfotransferases by an integrated biosynthetic and crystallographic approach. (United States)

    Liu, Jian; Moon, Andrea F; Sheng, Juzheng; Pedersen, Lars C


    Heparan sulfates (HSs) have potential therapeutic value as anti-inflammatory and antimetastasis drugs, in addition to their current use as anticoagulants. Recent advances in chemoenzymatic synthesis of HS provide a way to conveniently produce homogenous HS with different biological properties. Crystal structures of sulfotransferases involved in this process are providing atomic detail of their substrate binding clefts and interactions with their HS substrates. In theory, the flexibility of this method can be increased by modifying the specificities of the sulfotransferases based on the structures, thereby producing a new array of products.

  20. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A., E-mail: [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom)


    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  1. Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF family: improved signature and prediction of substrate specificity

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    Jeandroz Sylvain


    Full Text Available Abstract Background The Cation Diffusion Facilitator (CDF family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters. Results Representative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed. Conclusion In the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be

  2. Molecular and enzymatic properties of a cathepsin L-like proteinase with distinct substrate specificity from northern shrimp (Pandalus borealis). (United States)

    Aoki, H; Ahsan, M N; Watabe, S


    We purified a cathepsin L-like proteinase to homogeneity from the hepatopancreas of northern shrimp Pandalus borealis by several chromatographic procedures. The purified proteinase showed the highest specificity for leucine residue at P2, a specificity pattern similar to cathepsins S and K whereas proline and arginine residues were not suitable as P2 substrates. However, unlike these proteinases, it accepted valine almost equally to the phenylalanine residue at P2. The shrimp cathepsin was strongly inhibited by E-64, leupeptin and antipain, while benzyloxycarbonyl-Phe-Tyr(t-Bu)-CHN2, a specific inhibitor of cathepsin L, remained largely ineffective. Next, we determined the primary structure of the shrimp enzyme by molecular cloning and investigated the residues constituting the S2 subsite, which is possibly involved in its unusual substrate specificity. The deduced amino acid sequence of the shrimp proteinase shared the highest identity of 65% with a cathepsin L-like proteinase from lobster, but its identity to the well-characterized mammalian cathepsins S, L, and K fell within narrower ranges of 52-55%. However, the shrimp proteinase differed from these cathepsins in some key residues including, for example, the unique occurrence of cysteine and glutamine residues at the structurally important S2 subsite. Interestingly, transcripts of this proteinase were exclusively detected in the shrimp gut coinciding with its broad pH activity and stability profiles, which is also unusual as a cysteine proteinase. These results suggest that the shrimp enzyme is homologous to mammalian cathepsins S, L, and K, but is distinct from each of these proteinases in both enzymatic and structural properties.

  3. Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei. (United States)

    Yang, Shaoqing; Qin, Zhen; Duan, Xiaojie; Yan, Qiaojuan; Jiang, Zhengqiang


    Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2-C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi. The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases.

  4. Specific Heat of Hollow Nanosphere Coupled to Substrate: Quantum Size Effects

    Institute of Scientific and Technical Information of China (English)


    With the help of the elastic wave theory, in the perturbed approximation the density-of-states for vibrational modes and the specific heat axe studied for different hollow Si nanospheres, coupled with a semi-infinite substrate. We find that the modes of such coupled hollow spheres are significantly broadened and shifted toward low frequencies. The specific heat of the coupled hollow nanosphere is bigger than an isolated one due to the coupling interaction and quantum size effects. The predicted coupling and size enhancements on specific heat are probed in thermal experiments.

  5. Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus. (United States)

    Lukacin, Richard; Matern, Ulrich; Specker, Silvia; Vogt, Thomas


    Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

  6. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs (United States)

    Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.


    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

  7. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Directory of Open Access Journals (Sweden)

    Lalida Shank


    Full Text Available Peroxidases (POD and polyphenol oxidase (PPO are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from pea (PDB ID:1APX, whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X. Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

  8. Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities (United States)

    Kalupov, Timofey; Brillard-Bourdet, Michèle; Dadé, Sébastien; Serrano, Hélène; Wartelle, Julien; Guyot, Nicolas; Juliano, Luiz; Moreau, Thierry; Belaaouaj, Azzaq; Gauthier, Francis


    It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases. PMID:19833730

  9. A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance. (United States)

    Sandersjöö, Lisa; Kostallas, George; Löfblom, John; Samuelson, Patrik


    Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

  10. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve (United States)

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M. Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O.; Marasco, Wayne A.


    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies. PMID:27619409

  11. Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10.

    Directory of Open Access Journals (Sweden)

    Kathryn A K Finton


    Full Text Available The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.

  12. [Substrate specificity of Cryptococcus albidus and Eupenicillium erubescens alpha-L-rhamnosidases]. (United States)

    Hudzenko, O V; Varbanets', L D


    The substrate specificity of Cryptococcus albidus and Eupenicillium erubescens alpha-L-rhamnosidases has been investigated. It is shown that the enzymes are able to act on synthetic and natural substrates, such as naringin, neohesperidin. alpha-L-Rhamnosidases hydrolysed the latter ones very efficiently, in this case E. erubescens enzyme was characterized by higher values of V(max) in comparison with the enzyme of C. albidus. However the C. albidus alpha-L-rhamnosidase showed greater affinity for naringin and neohesperidin than the enzyme of E. erubescens (K(m) 0.77 and 3.3 mM and 5.0 and 3.0 mM, respectively). As regards the synthetic derivatives of monosaccharides, both enzymes exhibited narrow specificity for glycon: E. erubescens alpha-L-rhamnosidase--only to the p-nitrophenyl-alpha-L-rhamnopiranoside (K(m) 1.0 mM, V(max) 120 micromol/min/mg protein), and C. albidus--to p-nitrophenyl-alpha-D-glucopyranoside (K(m) 10 mM, V(max) 5 micromol/min/mg protein). Thus, it was found that the enzyme preparations of E. erubescens and C. albidus are differed by their substrate specificity. The ability of E. erubescens and C. albidus alpha-L-rhamnosidases to hydrolyse natural substrates: naringin and neohesperidin, evidences for their specificity for alpha-1,2-linked L-rhamnose. Based on these data, we can predict the use of E. erubescens and C. albidus alpha-L-rhamnosidases in various industries, food industry in particular. This is also confirmed by the fact that the investigated alpha-L-rhamnosidases were stable at 20% concentration of ethanol and 500 mM glucose in the reaction mixture.

  13. Substrate specificity of platypus venom L-to-D-peptide isomerase. (United States)

    Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W


    The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

  14. Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity. (United States)

    Welin, Martin; Wang, Liya; Eriksson, Staffan; Eklund, Hans


    Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

  15. The substrate specificities of sunflower and soybean phospholipases D using transphosphatidylation reaction

    Directory of Open Access Journals (Sweden)

    Abdelkafi Slim


    Full Text Available Abstract Background Phospholipase D (PLD belongs to a lipolytic enzyme subclass which catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond. Results In this work, we have studied the substrate specificity of PLDs from germinating sunflower seeds and cultured-soybean cells, using their capacity of transphosphatidylation. In the presence of a nucleophilic acceptor, such as [14C]ethanol, PLD catalyzes the production of phosphatidyl-[14C]-ethanol. The resulting product is easily identified since it is well separated from the other lipids by thin-layer chromatography. The main advantage of this assay is that the phospholipid used as substrate does not need to be radiolabelled and thus allow us a large choice of polar heads and fatty acids. In vitro, we observed that sunflower and soybean cell PLD show the following decreasing order of specificity: phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol; while phosphatidylserine and phosphatidylinositol are utilized much less efficiently. Conclusions The substrate specificity is modulated by the fatty acid composition of the phosphatidylcholine used as well as by the presence of other charged phospholipids.

  16. NMR-based screening method for transglutaminases: rapid analysis of their substrate specificities and reaction rates. (United States)

    Shimba, Nobuhisa; Yokoyama, Kei-ichi; Suzuki, Ei-ichiro


    Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins such as tofu, boiled fish paste, and sausage. Other transglutaminases (TGases) are expected to be used in the same way, and also to extend the scope of industrial applications to materials, drugs, and so on. The TGases have great diversity, not only in amino acid sequence and size, but also in their substrate specificities and catalytic activities, and therefore, it is quite difficult to estimate their reactivity. We have developed an NMR-based method using the enzymatic labeling technique (ELT) for simultaneous analysis of the substrate specificities and reaction rates of TGases. It is quite useful for comparing the existing TGases and for screening new TGases or TGases variants. This method has shown that MTG is superior for industrial use because of its lower substrate specificity compared with those of guinea pig liver transglutaminase (GTG) and red sea bream liver transglutaminase (FTG). We have also found that an MTG variant lacking an N-terminal aspartic acid residue has higher activity than that of the native enzyme.

  17. GSHSite: exploiting an iteratively statistical method to identify s-glutathionylation sites with substrate specificity.

    Directory of Open Access Journals (Sweden)

    Yi-Ju Chen

    Full Text Available S-glutathionylation, the covalent attachment of a glutathione (GSH to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA. TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN and human protein tyrosine phosphatase 1b (PTP1B. Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (, for identifying uncharacterized GSH substrate sites on the protein sequences.

  18. Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.

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    Martin Mahro

    Full Text Available In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3. The sequence alignment of different aldehyde oxidase (AOX isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR. Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.

  19. Amino acid residues important for substrate specificity of the amino acid permeases Can I p and Gnp I p in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Kielland-Brandt, M.C.


    Deletion of the general amino acid permease gene GAP1 abolishes uptake of L-citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations...... in the arginine permease gene CAN1. One similar mutation was found in the glutamine-asparagine permease gene GNP1. L-[C-14]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L-[C-14......]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations affect two proline residues (P148 and P313) that are both conserved in the amino acid-polyamine-choline (APC) transporter superfamily. We found mutations...

  20. The multi-protein family of sulfotransferases in plants: Composition, occurrence, substrate specificity and functions

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    Felix eHirschmann


    Full Text Available All members of the sulfotransferase (SOT, EC 2.8.2.- protein family transfer a sulfuryl group from the donor 3´-phosphoadenosine 5´-phosphosulfate (PAPS to an appropriate hydroxyl group of several classes of substrates. The primary structure of these enzymes is characterized by a histidine residue in the active site, defined PAPS binding sites and a longer SOT domain. Proteins with this SOT domain occur in all organisms from all three domains, usually as a multi-protein family. Arabidopsis thaliana SOTs, the best characterized SOT multi-protein family, contains 21 members. The substrates for several plant enzymes have already been identified, such as glucosinolates, brassinosteroids, jasmonates, flavonoids, and salicylic acid. Much information has been gathered on desulfo-glucosinolate (dsGl SOTs in A. thaliana. The three cytosolic dsGl SOTs show slightly different expression patterns. The recombinant proteins reveal differences in their affinity to indolic and aliphatic dsGls. Also the respective recombinant dsGl SOTs from different A. thaliana ecotypes differ in their kinetic properties. However, determinants of substrate specificity and the exact reaction mechanism still need to be clarified. Probably, the three-dimensional structures of more plant proteins need to be solved to analyze the mode of action and the responsible amino acids for substrate binding. In addition to A. thaliana, more plant species from several families need to be investigated to fully elucidate the diversity of sulfated molecules and the way of biosynthesis catalyzed by SOT enzymes.

  1. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J


    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  2. 2.0A resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine: substrate specificity and molecular motions related to substrate binding. (United States)

    Andersen, Ole Andreas; Stokka, Anne J; Flatmark, Torgeir; Hough, Edward


    The crystal structures of the catalytic domain of human phenylalanine hydroxylase (hPheOH) in complex with the physiological cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the substrate analogues 3-(2-thienyl)-L-alanine (THA) or L-norleucine (NLE) have been determined at 2.0A resolution. The ternary THA complex confirms a previous 2.5A structure, and the ternary NLE complex shows that similar large conformational changes occur on binding of NLE as those observed for THA. Both structures demonstrate that substrate binding triggers structural changes throughout the entire protomer, including the displacement of Tyr138 from a surface position to a buried position at the active site, with a maximum displacement of 20.7A for its hydroxyl group. Two hinge-bending regions, centred at Leu197 and Asn223, act in consort upon substrate binding to create further large structural changes for parts of the C terminus. Thus, THA/L-Phe binding to the active site is likely to represent the epicentre of the global conformational changes observed in the full-length tetrameric enzyme. The carboxyl and amino groups of THA and NLE are positioned identically in the two structures, supporting the conclusion that these groups are of key importance in substrate binding, thus explaining the broad non-physiological substrate specificity observed for artificially activated forms of the enzyme. However, the specific activity with NLE as the substrate was only about 5% of that with THA, which is explained by the different affinities of binding and different catalytic turnover.

  3. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    Full Text Available Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

  4. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin. (United States)

    Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R


    Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

  5. Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments. (United States)

    Crits-Christoph, Alexander; Robinson, Courtney K; Ma, Bing; Ravel, Jacques; Wierzchos, Jacek; Ascaso, Carmen; Artieda, Octavio; Souza-Egipsy, Virginia; Casero, M Cristina; DiRuggiero, Jocelyne


    Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates

  6. Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in hyper-arid environments

    Directory of Open Access Journals (Sweden)

    Alexander eCrits-Christoph


    Full Text Available Under extreme water deficit, endolithic (inside rock microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP and polyketides (PK. Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both

  7. Using bacteria to determine protein kinase specificity and predict target substrates.

    Directory of Open Access Journals (Sweden)

    Michael F Chou

    Full Text Available The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A and acidophilic (Casein Kinase II kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

  8. No significant difference in antigenicity or tissue transglutaminase substrate specificity of Irish and US wheat gliadins. (United States)

    Keaveny, A P; Offner, G D; Bootle, E; Nunes, D P


    The prevalence of clinical celiac disease has been shown to vary both across time and between genetically similar populations. Differences in wheat antigenicity and transglutaminase substrate properties are a possible explanation for these differences. This study assessed the antigenicity and transglutaminase substrate specificities of gliadins from regions of high and low celiac disease prevalence. Gliadin was extracted from three commercial US wheat sources and two Irish sources. SDS-PAGE and western blotting revealed minor, but significant variations in the gliadin extracts. However, ELISA showed no difference in the antigenicity of these gliadins. Transglutaminase pretreatment of gliadin resulted in no significant change in gliadin antigenicity and kinetic studies showed that the Kms of the various gliadins were very similar. Purified IgA and IgG had no effect on transglutaminase activity. In summary, minor variations in wheat gliadins are unlikely to explain the observed differences in disease expression across genetically similar populations.

  9. Molecular mechanism of substrate specificity in the bacterial neutral amino acid transporter LeuT. (United States)

    Noskov, Sergei Y


    The recently published X-ray structure of LeuT, a Na(+)/Cl(-)-dependent neurotransmitter transporter, has provided fresh impetus to efforts directed at understanding the molecular principles governing specific neurotransmitter transport. The combination of the LeuT crystal structure with the results of molecular simulations enables the functional data on specific binding and transport to be related to molecular structure. All-atom FEP and molecular dynamics (MD) simulations of LeuT embedded in an explicit membrane were performed alongside a decomposition analysis to dissect the molecular determinants of the substrate specificity of LeuT. It was found that the ligand must be in a zwitterionic (ZW) form to bind tightly to the transporter. The theoretical results on the absolute binding-free energies for leucine, alanine, and glycine show that alanine can be a potent substrate for LeuT, although leucine is preferred, which is consistent with the recent experimental data (Singh et al., Nature 2007;448:952-956). Furthermore, LeuT displays robust specificity for leucine over glycine. Interestingly, the ability of LeuT to discriminate between substrates relies on the dynamics of residues that form its binding pocket (e.g., F253 and Q250) and the charged side chains (R30-D404) from a second coordination shell. The water-mediated R30-D404 salt bridge is thought to be part of the extracellular (EC) gate of LeuT. The introduction of a polar ligand such as glycine to the water-depleted binding pocket of LeuT gives rise to structural rearrangements of the R30-D404-Q250 hydrogen-bonding network and leads to increased hydration of the binding pocket. Conformational changes associated with the broken hydrogen bond between Q250 and R30 are shown to be important for tight and selective ligand binding to LeuT.

  10. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Directory of Open Access Journals (Sweden)

    Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike


    Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  11. Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities. (United States)

    Osuji, Godson O; Braithwaite, Cleantis; Fordjour, Kusi; Madu, Wenceslaus C; Beyene, Asefa; Roberts, Paul S; Wright, Victor


    Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

  12. Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families (United States)

    Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin


    Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

  13. On the substrate specificity of the rice strigolactone biosynthesis enzyme DWARF27

    KAUST Repository

    Bruno, Mark


    Main conclusion: The β-carotene isomerase OsDWARF27 is stereo- and double bond-specific. It converts bicyclic carotenoids with at least one unsubstituted β-ionone ring. OsDWARF27 may contribute to the formation of α-carotene-based strigolactone-like compounds.Strigolactones (SLs) are synthesized from all-trans-β-carotene via a pathway involving the β-carotene isomerase DWARF27, the carotenoid cleavage dioxygenases 7 and 8 (CCD7, CCD8), and cytochrome P450 enzymes from the 711 clade (MAX1 in Arabidopsis). The rice enzyme DWARF27 was shown to catalyze the reversible isomerization of all-trans- into 9-cis-β-carotene in vitro. β-carotene occurs in different cis-isomeric forms, and plants accumulate other carotenoids, which may be substrates of DWARF27. Here, we investigated the stereo and substrate specificity of the rice enzyme DWARF27 in carotenoid-accumulating E. coli strains and in in vitro assays performed with heterologously expressed and purified enzyme. Our results suggest that OsDWARF27 is strictly double bond-specific, solely targeting the C9–C10 double bond. OsDWARF27 did not introduce a 9-cis-double bond in 13-cis- or 15-cis-β-carotene. Substrates isomerized by OsDWARF27 are bicyclic carotenoids, including β-, α-carotene and β,β-cryptoxanthin, that contain at least one unsubstituted β-ionone ring. Accordingly, OsDWARF27 did not produce the abscisic acid precursors 9-cis-violaxanthin or -neoxanthin from the corresponding all-trans-isomers, excluding a direct role in the formation of this carotenoid derived hormone. The conversion of all-trans-α-carotene yielded two different isomers, including 9′-cis-α-carotene that might be the precursor of strigolactones with an ε-ionone ring, such as the recently identified heliolactone. © 2016 Springer-Verlag Berlin Heidelberg

  14. Substrate specificity and molecular modelling of the feline herpesvirus-1 thymidine kinase. (United States)

    Hussein, Islam T M; Miguel, Ricardo Núñez; Tiley, Laurence S; Field, Hugh J


    Feline herpesvirus-1 (FHV-1) causes a severe upper respiratory and ocular disease in cats. An effective antiviral compound is required for treating FHV-1 infections. The virus-encoded thymidine kinase (TK) is the molecular basis for selective activation of commonly used antiviral nucleoside analogue drugs, e.g. acyclovir (ACV), penciclovir (PCV) and ganciclovir (GCV). The substrate specificity of a recombinant FHV-1 TK, expressed in Escherichia coli, was studied. FHV-1 TK efficiently phosphorylated its natural substrate deoxythymidine. However, it exhibited relatively lower affinity for the guanosine analogue substrates. PCV was most efficiently phosphorylated, followed by GCV, with approximately twofold reduction in the phosphorylation rate. The lowest phosphorylation rate was recorded for ACV. To correlate these biochemical data with structural features of the FHV-1 TK, a three-dimensional (3D) model of this enzyme was constructed based on sequence homology with two other herpesviral TKs, encoded by equine herpesvirus-4 (EHV-4) and herpes simplex-1 (HSV-1). Mutational analysis of the amino acids forming the FHV-1 TK active site identified two residues (Y29 and F144) as being critical for the differential ability of this enzyme to phosphorylate nucleoside analogues. A double substitution of Y29H/F144Y resulted in a threefold increase in the ACV phosphorylation rate.

  15. An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

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    Vermeulen Michiel


    Full Text Available Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

  16. Dynamic changes in the secondary structure of ECE-1 and XCE account for their different substrate specificities

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    Ul-Haq Zaheer


    Full Text Available Abstract Background X-converting enzyme (XCE involved in nervous control of respiration, is a member of the M13 family of zinc peptidases, for which no natural substrate has been identified yet. In contrast, it’s well characterized homologue endothelin-converting enzyme-1 (ECE-1 showed broad substrate specificity and acts as endopeptidase as well as dipeptidase. To explore the structural differences between XCE and ECE-1, homology model of XCE was built using the complex structure of ECE-1 with phosphoramidon (pdb-id: 3DWB as template. Phosphoramidon was docked into the binding site of XCE whereas phosphate oxygen of the inhibitor was used as water molecule to design the apo forms of both enzymes. Molecular dynamics simulation of both enzymes was performed to analyze the dynamic nature of their active site residues in the absence and presence of the inhibitor. Results Homology model of XCE explained the role of non-conserved residues of its S2’ subsite. Molecular dynamics (MD simulations identified the flexible transitions of F149/I150, N566/N571, W714/W719, and R145/R723 residues of ECE-1/XCE for the strong binding of the inhibitor. Secondary structure calculations using DSSP method reveals the folding of R145/R723 residue of ECE-1/XCE into β-sheet structure while unfolding of the S2’ subsite residues in aECE-1 and sustained compact folding of that of aXCE. The results evaluated are in good agreement with available experimental data, thus providing detailed molecular models which can explain the structural and specificities differences between both zinc peptidases. Conclusions Secondary structure changes of both enzymes during the simulation time revealed the importance of β-sheet structure of R145/R723 for its binding with the terminal carboxylate group of the inhibitor. Unfolding of the α-helix comprising the S2’ subsite residues in aECE-1 correlate well with its endopeptidase activity while their compact folding in aXCE may

  17. Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2)


    Kennedy, David J.; Gatfield, Kelly M.; Winpenny, John P; Ganapathy, Vadivel; Thwaites, David T.


    Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrate-evoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2.Amino (and imino)...

  18. Substrate specificity of low-molecular mass bacterial DD-peptidases. (United States)

    Nemmara, Venkatesh V; Dzhekieva, Liudmila; Sarkar, Kumar Subarno; Adediran, S A; Duez, Colette; Nicholas, Robert A; Pratt, R F


    The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD

  19. The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II. (United States)

    Sivaraman, Sharada; Kirsch, Jack F


    Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.

  20. Carotenoids in Rhodoplanes species: variation of compositions and substrate specificity of predicted carotenogenesis enzymes. (United States)

    Takaichi, Shinichi; Sasikala, Ch; Ramana, Ch V; Okamura, Keiko; Hiraishi, Akira


    Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.

  1. Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase. (United States)

    Liu, Chunhui; Sheng, Juzheng; Krahn, Juno M; Perera, Lalith; Xu, Yongmei; Hsieh, Po-Hung; Dou, Wenfang; Liu, Jian; Pedersen, Lars C


    Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.

  2. Structural basis for substrate specificity of alphavirus nsP2 proteases. (United States)

    Russo, Andrew T; Malmstrom, Robert D; White, Mark A; Watowich, Stanley J


    The alphavirus nsP2 protease is essential for correct processing of the alphavirus nonstructural polyprotein (nsP1234) and replication of the viral genome. We have combined molecular dynamics simulations with our structural studies to reveal features of the nsP2 protease catalytic site and S1'-S4 subsites that regulate the specificity of the protease. The catalytic mechanism of the nsP2 protease appears similar to the papain-like cysteine proteases, with the conserved catalytic dyad forming a thiolate-imidazolium ion pair in the nsP2-activated state. Substrate binding likely stabilizes this ion pair. Analysis of bimolecular complexes of Venezuelan equine encephalitis virus (VEEV) nsP2 protease with each of the nsP1234 cleavage sites identified protease residues His(510), Ser(511), His(546) and Lys(706) as critical for cleavage site recognition. Homology modelling and molecular dynamics simulations of diverse alphaviruses and their cognate cleavage site sequences revealed general features of substrate recognition that operate across alphavirus strains as well as strain specific covariance between binding site and cleavage site residues. For instance, compensatory changes occurred in the P3 and S3 subsite residues to maintain energetically favourable complementary binding surfaces. These results help explain how alphavirus nsP2 proteases recognize different cleavage sites within the nonstructural polyprotein and discriminate between closely related cleavage targets.

  3. The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities. (United States)

    Marelja, Zvonimir; Dambowsky, Miriam; Bolis, Marco; Georgiou, Marina L; Garattini, Enrico; Missirlis, Fanis; Leimkühler, Silke


    In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.

  4. High-throughput profiling of N-myristoylation substrate specificity across species including pathogens. (United States)

    Traverso, José A; Giglione, Carmela; Meinnel, Thierry


    One of the most critical modifications affecting the N-terminus of proteins is N-myristoylation. This irreversible modification affects the membrane-binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N-myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state-of-the-art proteomics approaches or bioinformatics tools. A large part of N-myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high-throughput profiling protein N-myristoylation specificity. We show that myristoylation predictive tools need to be fine-tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.

  5. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition. (United States)

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E


    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.

  6. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis

    DEFF Research Database (Denmark)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram;


    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr...... and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual...... isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide...

  7. Global, Broad, or Specific Cognitive Differences? Using a MIMIC Model to Examine Differences in CHC Abilities in Children with Learning Disabilities (United States)

    Niileksela, Christopher R.; Reynolds, Matthew R.


    This study was designed to better understand the relations between learning disabilities and different levels of latent cognitive abilities, including general intelligence (g), broad cognitive abilities, and specific abilities based on the Cattell-Horn-Carroll theory of intelligence (CHC theory). Data from the "Differential Ability…

  8. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues (United States)

    This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from s...

  9. Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4

    DEFF Research Database (Denmark)

    Sternberg, Claus; Eberl, Leo; Sanchezromero, Juan M.


    The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly ...

  10. Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme. (United States)

    Vizitiu, D; Kriste, A G; Campbell, A S; Thatcher, G R


    Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.

  11. The RSC Chromatin Remodeling Complex Bears an Essential Fungal-Specific Protein Module With Broad Functional Roles


    Wilson, Boris; Erdjument-Bromage, Hediye; Tempst, Paul; Bradley R Cairns


    RSC is an essential and abundant ATP-dependent chromatin remodeling complex from Saccharomyces cerevisiae. Here we show that the RSC components Rsc7/Npl6 and Rsc14/Ldb7 interact physically and/or functionally with Rsc3, Rsc30, and Htl1 to form a module important for a broad range of RSC functions. A strain lacking Rsc7 fails to properly assemble RSC, which confers sensitivity to temperature and to agents that cause DNA damage, microtubule depolymerization, or cell wall stress (likely via tran...

  12. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus


    , the functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...... bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized...

  13. A structural account of substrate and inhibitor specificity differences between two Naphthol reductases

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Thompson, J.E.; Fahnestock, S.; Valent, B.; Jordan, D.B. (DuPont)


    Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 {angstrom} resolution structure allows for comparisons with the 1.7 {angstrom} resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH{sub 2} groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

  14. Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase. (United States)

    Petković, Hrvoje; Sandmann, Axel; Challis, Iain R; Hecht, Hans-Jürgen; Silakowski, Barbara; Low, Lindsey; Beeston, Nicola; Kuscer, Enej; Garcia-Bernardo, Jose; Leadlay, Peter F; Kendrew, Steven G; Wilkinson, Barrie; Müller, Rolf


    The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.

  15. High specific surface gold electrode on polystyrene substrate: Characterization and application as DNA biosensor. (United States)

    Yang, Zhiliu; Liu, Yichen; Lu, Wei; Yuan, Qingpan; Wang, Wei; Pu, Qiaosheng; Yao, Bo


    In the past decades, many efforts have been made to improve the sensitivity and specificity of electrochemical DNA biosensors. However, it is still strongly required to develop disposable and reliable DNA biosensors for wide and practical application. In this article, we reported superior electrochemical properties of an integrated plastic-gold electrode (PGE) fabricated in-house by chemical plating on polystyrene substrate. PGEs were found having extremely high capacity of DNA immobilization compared with gold electrodes fabricated by standard sputtering based photolithography. Unique nano-structured surface was observed on PGEs through morphology techniques, which would to some extend give an explanation to higher capacity of DNA immobilization on PGEs. A probable mechanism of carboxylic acid produced on polystyrene substrate after exposure to UV irradiation was proposed and discussed for the first time. This biosensor was applied to detection and manipulate of DNA hybridization. Detection limit of 7.2×10(-11) M and 1-500 nM of linearity range was obtained.

  16. [Microbial alpha-amylases: physicochemical properties, substrate specificity and domain structure]. (United States)

    Avdiiuk, K V; Varbanets', L D


    The current literature data on producers, physico-chemical properties and substrate specificity of a-amylases produced by microbes from different taxonomic groups such as bacteria, fungi and yeasts are discussed in the survey. Synthesis of alpha-amylase majority is an inducible process which is stimulated in the presence of starch or products of its hydrolysis. It is possible to increase enzymes activity level by optimization of cultivation conditions of strains-producers. alpha-Amylases, isolated from different sources are distinguished in their physico-chemical properties, particularly in their molecular weights, pH- and thermooptimums, inhibitors and activators. The enzymes hydrolyse soluble starch, amylose, amylopectin, glycogen, maltodextrins, alpha- and beta3-cyclodextrins and other carbohydrate substrates. It is well known that alpha-amylases belong to GH-13 family of glycosyl-hydrolases, which contain the catalytic domain A as (beta/alpha)8-barrel. In addition to domain A, alpha-amylases contain two other domains: B and C, which are localized approximately on opposite sides of (beta/alpha)8-barrel. Most of the known alpha-amylases contain calcium ion, which is located on the surface between domains A and B and plays an important role in stability and activity of the enzyme.

  17. Combining substrate specificity analysis with support vector classifiers reveals feruloyl esterase as a phylogenetically informative protein group

    DEFF Research Database (Denmark)

    Olivares Hernandez, Roberto; Sunner, Hampus; Frisvad, Jens Christian;


    predict the taxonomic Class (~86% correct classification), Order (~88% correct classification) and Family (~88% correct classification) that the 34 Ascomycota belong to, using the activity profiles of the FAEs. Conclusion/Significance The good correlation with the FAEs substrate specificities that we have...... the relative activity of FAEs against a variety of model substrates as a novel predictive tool for Ascomycota taxonomic classification. Our approach consists of two analytical steps; (1) an initial unsupervised analysis to cluster the FAEs substrate specificity data which were generated by cultivation of 34...

  18. Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability. (United States)

    Svensson, B


    Most starch hydrolases and related enzymes belong to the alpha-amylase family which contains a characteristic catalytic (beta/alpha)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the alpha-amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the alpha-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the beta-->alpha loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of alpha-amylases, changed the distribution of alpha-, beta- and gamma-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative alpha-1,4:alpha-1,6 dual-bond specificity of neopullulanase. Barley alpha-amylase isozyme hybrids and Bacillus alpha-amylases demonstrate the impact of a small domain B protruding from the (beta/alpha)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as alpha-amylase, starch branching and debranching enzymes, and amylomaltase.

  19. Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

    Energy Technology Data Exchange (ETDEWEB)

    Adhikary, Suraj; Eichman, Brandt F. (Vanderbilt)


    DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

  20. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  1. Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit (United States)

    Tatebe, Hisashi; Murayama, Shinichi; Yonekura, Toshiya; Hatano, Tomoyuki; Richter, David; Furuya, Tomomi; Kataoka, Saori; Furuita, Kyoko; Kojima, Chojiro; Shiozaki, Kazuhiro


    The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT. DOI: PMID:28264193

  2. Substrate specificity and ion coupling in the Na+/betaine symporter BetP. (United States)

    Perez, Camilo; Koshy, Caroline; Ressl, Susanne; Nicklisch, Sascha; Krämer, Reinhard; Ziegler, Christine


    BetP is an Na(+)-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT-fold) that BetP shares with other sequence-unrelated Na(+)-coupled symporters. Numerous structures of LeuT-fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and co-transported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a single-point mutation--glycine to aspartate--into an H(+)-coupled choline-specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP-G153D demonstrates a new inward-facing open conformation for BetP. Choline binding to a location close to the second, low-affinity sodium-binding site (Na2) of LeuT-fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cation-binding site in BetP, playing a key role in a proposed molecular mechanism of Na(+) and H(+) coupling in BCC transporters.

  3. Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae. (United States)

    Lukacin, Richard; Schreiner, Stephan; Silber, Katrin; Matern, Ulrich


    Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.

  4. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Caitlin E. Mullarkey


    Full Text Available Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS, we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  5. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner. (United States)

    Mullarkey, Caitlin E; Bailey, Mark J; Golubeva, Diana A; Tan, Gene S; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E; Bowdish, Dawn M; Miller, Matthew S; Palese, Peter


    Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  6. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)


    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  7. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. (United States)

    Julien, Olivier; Zhuang, Min; Wiita, Arun P; O'Donoghue, Anthony J; Knudsen, Giselle M; Craik, Charles S; Wells, James A


    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

  8. Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Bale, Shridhar; Baba, Kavita; McCloskey, Diane E.; Pegg, Anthony E.; Ealick, Steven E.


    The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5{prime}-deoxy-5{prime}-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

  9. Substrate-specific gene expression in Batrachochytrium dendrobatidis, the chytrid pathogen of amphibians.

    Directory of Open Access Journals (Sweden)

    Erica Bree Rosenblum

    Full Text Available Determining the mechanisms of host-pathogen interaction is critical for understanding and mitigating infectious disease. Mechanisms of fungal pathogenicity are of particular interest given the recent outbreaks of fungal diseases in wildlife populations. Our study focuses on Batrachochytrium dendrobatidis (Bd, the chytrid pathogen responsible for amphibian declines around the world. Previous studies have hypothesized a role for several specific families of secreted proteases as pathogenicity factors in Bd, but the expression of these genes has only been evaluated in laboratory growth conditions. Here we conduct a genome-wide study of Bd gene expression under two different nutrient conditions. We compare Bd gene expression profiles in standard laboratory growth media and in pulverized host tissue (i.e., frog skin. A large proportion of genes in the Bd genome show increased expression when grown in host tissue, indicating the importance of studying pathogens on host substrate. A number of gene classes show particularly high levels of expression in host tissue, including three families of secreted proteases (metallo-, serine- and aspartyl-proteases, adhesion genes, lipase-3 encoding genes, and a group of phylogenetically unusual crinkler-like effectors. We discuss the roles of these different genes as putative pathogenicity factors and discuss what they can teach us about Bd's metabolic targets, host invasion, and pathogenesis.

  10. Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates. (United States)

    Andersen, Christian; Maier, Elke; Kemmer, Gabrielle; Blass, Julia; Hilpert, Anna-Karina; Benz, Roland; Reidl, Joachim


    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.

  11. Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Xiao; Chen, Ming; Zhang, Sheng; Yu, Zhi-Hong; Sun, Jin-Peng; Wang, Lina; Liu, Sijiu; Imasaki, Tsuyoshi; Takagi, Yuichiro; Zhang, Zhong-Yin (Indiana-Med)


    A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an 'inverse alanine scanning' combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.

  12. Sortase A substrate specificity in GBS pilus 2a cell wall anchoring. (United States)

    Necchi, Francesca; Nardi-Dei, Vincenzo; Biagini, Massimiliano; Assfalg, Michael; Nuccitelli, Annalisa; Cozzi, Roberta; Norais, Nathalie; Telford, John L; Rinaudo, C Daniela; Grandi, Guido; Maione, Domenico


    Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

  13. Sortase A substrate specificity in GBS pilus 2a cell wall anchoring.

    Directory of Open Access Journals (Sweden)

    Francesca Necchi

    Full Text Available Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS, is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b, whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA, whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2 is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40 able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein. Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

  14. Comparing the glucuronidation capacity of the feline liver with substrate-specific glucuronidation in dogs

    NARCIS (Netherlands)

    van Beusekom, C D; Fink-Gremmels, J; Schrickx, J A


    This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP-glucuronosyltransferases (UGTs). To this end, Michaelis-Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were compar

  15. The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

    Energy Technology Data Exchange (ETDEWEB)

    Khadempour, Lily [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Zoology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA; Burnum-Johnson, Kristin E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Baker, Erin S. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Nicora, Carrie D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Webb-Robertson, Bobbie-Jo M. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; White, Richard A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Huang, Eric L. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Currie, Cameron R. [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA


    Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles were significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.

  16. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases. (United States)

    Cha, Hyung Jin; Jeong, Jae-Hee; Rojviriya, Catleya; Kim, Yeon-Gil


    YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

  17. Substrate-specific modifications on magnetic iron oxide nanoparticles as an artificial peroxidase for improving sensitivity in glucose detection

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yanping; Yu Faquan, E-mail: [Key Laboratory for Green Chemical Process of Ministry of Education, College of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073 (China)


    Magnetic iron oxide nanoparticles (MION) were recently found to act as a peroxidase with intrinsic advantages over natural counterparts. Their limited affinity toward catalysis substrates, however, dramatically reduces their utility. In this paper, some effective groups were screened out and conjugated on MION as substrate-specific modifications for improving MION's affinity to substrates and hence utility. Nanoparticles of four different superficial structures were synthesized and characterized by TEM, size, zeta potential and SQUID, and assayed for peroxidase activity. Glucose detection was selected as an application model system to evaluate the bonus thereof. Catalysis was found to follow Michaelis-Menten kinetics. Sulfhydryl groups incorporated on MION (SH-MION) notably improve the affinity toward a substrate (hydrogen peroxide) and so do amino groups (NH{sub 2}-MION) toward another substrate, proved by variation in the determined kinetic parameters. A synergistically positive effect was observed and an apparently elevated detection sensitivity and a significantly lowered detection limit of glucose were achieved when integrated with both sulfhydryl and amino groups (SH-NH{sub 2}-MION). Our findings suggest that substrate-specific surface modifications are a straightforward and robust strategy to improve MION peroxidase-like activity. The high activity extends magnetic nanoparticles to wide applications other than glucose detection.

  18. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases.

    Directory of Open Access Journals (Sweden)

    Hyung Jin Cha

    Full Text Available YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

  19. [Use of kenaf fibre in the elaboration of specific substrates for Pleurotus ostreatus (Jacq. ex Fr.) Kummer cultivation]. (United States)

    Pardo Giménez, Arturo; Perona Zamora, Ma Aquilina; Pardo Núñez, José


    In this study, the viability of the kenaf fibre use, alone or combined with cereal straw, vine shoots and olive mill dried waste, in the elaboration of specific substrates for the cultivation of Pleurotus ostreatus (Jacq. ex Fr.) Kummer, second mushroom in importance cultivated in Spain, is described. Furthermore, three different methods of preparation of the substrate have been considered in order to obtain selectivity for the growth and later fruiting of Pleurotus sporophore. As for the production parameters, the best results have been provided by the substrates that combined kenaf with straw and with vine shoots, being unfavourable the substrates based in just kenaf or combined with olive mill dried waste. As for the treatment applied to the materials, the immersion in water alone and subsequent pasteurization and thermophilic conditioning, together with the semi-anaerobic fermentation, has been favoured in front of the immersion in water with fungicide and later pasteurization.

  20. Substrate specifity and inhibitors of polyphenol oxidase in aspect of darkening of fresh and frozen mushrooms (Agaricus bisporus (Lange Sing.

    Directory of Open Access Journals (Sweden)

    Janusz Czapski


    Full Text Available Activity of mushroom polyphenol oxidase (PPO toward 6 substrates and inhibitory effect of cysteine, 2-mercaptoethanol, benzoic acid and sodium metabisulphite were determined. The o-diphenols which appeared to be the best substrates were: catechin, DOPA (L-3,4-dihydro-xyphenylalanine and chlorogcnic acid. Affinity of PPO crude preparation substrates to enzyme, expressed as inverse value of Michaelis constant was lower then affinity of catechol. Inhibitory effect depended on specifity of inhibitors and their concentration. Electrophoretic patterns of PPO of mushrooms reveals slow and fast moving 4 isoforms when DOPA was used as a substrate, 2 bands for catechin and chlorogenic acid while only one band showed activity toward tyrosine and p-cresol.

  1. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

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    Cédric Grauffel

    Full Text Available Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs. In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p. To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG, hNaa10p/AcCoA/substrate (substrate=MLG, EEE. Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic, hNaa20p (hydrophobic/basic, hNaa30p (basic and hNaa50p (hydrophobic. We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide

  2. Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Luc Dedieu

    Full Text Available The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

  3. Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G (United States)

    Larimer, Cassandra M.; Slavnic, Dejan; Pitstick, Lenore D.; Green, Jacalyn M.


    Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in

  4. Structural and mutational studies on substrate specificity and catalysis of Salmonella typhimurium D-cysteine desulfhydrase.

    Directory of Open Access Journals (Sweden)

    Sakshibeedu R Bharath

    Full Text Available Salmonella typhimurium DCyD (StDCyD is a fold type II pyridoxal 5' phosphate (PLP-dependent enzyme that catalyzes the degradation of D-Cys to H(2S and pyruvate. It also efficiently degrades β-chloro-D-alanine (βCDA. D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC. Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, βCDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS and L-cycloserine (LCS at resolutions ranging from 1.7 to 2.6 Å. The polypeptide fold of StDCyD consisting of a small domain (residues 48-161 and a large domain (residues 1-47 and 162-328 resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.

  5. Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal. (United States)

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre


    A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry.

  6. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    impaired mutant enzyme Y380A is described, in an effort to determine the potential role of SBS2 in amylopectin degradation. Progress curves of amylopectin degradation was best described by a bi-exponential equation comprising two rate constants (for reaction 'a' and 'b') associated with the degradation...... mechanism, in which the metal ion must bind prior to the substrate. Ni2+ and Cu2+ were inhibitors of ManA, which probably bind to the active site, but result in an enzyme unable to facilitate substrate binding. Numerous glycosidases have a carbohydrate binding domain (CBM) appended to the catalytic domain....... CBMs are predominantly thought to maintain the enzyme on its substrate to artificially increase the substrate concentration. Analogously, secondary binding sites (SBSs), which, contrary to CBMs are located on the catalytic domain, have been suggested to perform a similar task. Many putative SBSs have...

  7. Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541. (United States)

    Karabec, Martin; Łyskowski, Andrzej; Tauber, Katharina C; Steinkellner, Georg; Kroutil, Wolfgang; Grogan, Gideon; Gruber, Karl


    The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

  8. Substrate specific stimulation of NEIL1 by WRN but not the other human RecQ helicases

    DEFF Research Database (Denmark)

    Popuri, Venkateswarlu; Croteau, Deborah L; Bohr, Vilhelm A


    protein (WRN), one of the five human RecQ helicases, stimulates NEIL1 DNA glycosylase activity on oxidative DNA lesions. The goal of this study was to extend this observation and analyze the interaction between NEIL1 and all five human RecQ helicases. The DNA substrate specificity of the interaction...

  9. Structural insights into the specific recognition of N-heterocycle biodenitrogenation-derived substrates by microbial amide hydrolases. (United States)

    Wu, Geng; Chen, Duoduo; Tang, Hongzhi; Ren, Yiling; Chen, Qihua; Lv, Yang; Zhang, Zhenyi; Zhao, Yi-Lei; Yao, Yuxiang; Xu, Ping


    N-heterocyclic compounds from industrial wastes, including nicotine, are environmental pollutants or toxicants responsible for a variety of health problems. Microbial biodegradation is an attractive strategy for the removal of N-heterocyclic pollutants, during which carbon-nitrogen bonds in N-heterocycles are converted to amide bonds and subsequently severed by amide hydrolases. Previous studies have failed to clarify the molecular mechanism through which amide hydrolases selectively recognize diverse amide substrates and complete the biodenitrogenation process. In this study, structural, computational and enzymatic analyses showed how the N-formylmaleamate deformylase Nfo and the maleamate amidase Ami, two pivotal amide hydrolases in the nicotine catabolic pathway of Pseudomonas putida S16, specifically recognize their respective substrates. In addition, comparison of the α-β-α groups of amidases, which include Ami, pinpointed several subgroup-characteristic residues differentiating the two classes of amide substrates as containing either carboxylate groups or aromatic rings. Furthermore, this study reveals the molecular mechanism through which the specially tailored active sites of deformylases and amidases selectively recognize their unique substrates. Our work thus provides a thorough elucidation of the molecular mechanism through which amide hydrolases accomplish substrate-specific recognition in the microbial N-heterocycles biodenitrogenation pathway.

  10. Alteration of substrate specificities of thermophilic α/β hydrolases through domain swapping and domain interface optimization

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Zhou; Honglei Wang; Yuhang Zhang; Le Gao; Yan Feng


    Protein domain swapping is an efficient way in protein functional evolution in vivo and also has been proved to be an effective strategy to modify the function of the multidomain proteins in vitro.To explore the potentials of domain swapping for alteration of the enzyme substrate specificities and the structure-function relationship of the homologous proteins,here we constructed two chimeras from a pair of thermophilic members of the α/β hydrolase superfamily by grafting their functional domains to the conserved α/β hydrolase fold domain:a carboxylesterase from Archaeoglobus fulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyl chain lengths.We took two approaches to reduce the crossover disruptions when creating the chimeras:chose the residue which involved in the least contacts as the splicing site and optimized the newly formed domain interfaces of the chimeras by sitedirected mutations.Characterizations of AAM7 and PAR showed that these chimeras inherited the thermophilic property of both parents.In the aspect of substrate specificity,AAM7 and PAR showed highest activity towards short chain length substrate pNPC4 and middle chain length substrate pNPC8,similar to parent AFEST and apAPH,respectively.These results suggested that the substrate-binding domain is the dominant factor on enzyme substrate specificity,and the optimization of the newly formed domain interface is an important guarantee for successful domain swapping of proteins with low-sequence homology.

  11. Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase

    Energy Technology Data Exchange (ETDEWEB)

    Han,Q.; Gao, Y.; Robinson, H.; Li, J.


    Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant a-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate {beta}-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and {alpha}7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

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

    Directory of Open Access Journals (Sweden)

    Kishore Sarma


    Full Text Available 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.

  13. Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

    DEFF Research Database (Denmark)

    Welin, M.; Skovgaard, T.; Knecht, Wolfgang


    The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3...

  14. Short-term in-vitro expansion improves monitoring and allows affordable generation of virus-specific T-cells against several viruses for a broad clinical application.

    Directory of Open Access Journals (Sweden)

    René Geyeregger

    Full Text Available Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8(+ and CD4(+ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.

  15. Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK. (United States)

    Dias, Marcio V B; Huang, Fanglu; Chirgadze, Dimitri Y; Tosin, Manuela; Spiteller, Dieter; Dry, Emily F V; Leadlay, Peter F; Spencer, Jonathan B; Blundell, Tom L


    The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

  16. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.



    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  17. Functional analysis of glycoside hydrolase family 8 xylanases shows narrow but distinct substrate specificities and biotechnological potential. (United States)

    Pollet, Annick; Schoepe, Jan; Dornez, Emmie; Strelkov, Sergei V; Delcour, Jan A; Courtin, Christophe M


    The potential of glycoside hydrolase family (GH) 8 xylanases in biotechnological applications is virtually unexplored. Therefore, the substrate preference and hydrolysis product profiles of two GH8 xylanases were evaluated to investigate their activities and substrate specificities. A GH8 xylanase from an uncultured bacterium (rXyn8) shows endo action but very selectively releases xylotriose from its substrates. It has a higher activity than the Pseudoalteromonas haloplanktis GH8 endo-xylanase (PhXyl) on xylononaose and smaller xylo-oligosaccharides. PhXyl preferably degrades xylan substrates with a high degree of polymerization. It is sterically more hindered by arabinose substituents than rXyn8, producing larger end hydrolysis products. The specificities of rXyn8 and PhXyl differ completely from these of the previously described GH8 xylanases from Bifidobacterium adolescentis (BaRexA) and Bacillus halodurans (BhRex). As reducing-end xylose-releasing exo-oligoxylanases, they selectively release xylose from the reducing end of small xylo-oligosaccharides. The findings of this study show that GH8 xylanases have a narrow substrate specificity, but also one that strongly varies between family members and is distinct from that of GH10 and GH11 xylanases. Structural comparison of rXyn8, PhXyl, BaRexA, and BhRex showed that subtle amino acid changes in the glycon as well as the aglycon subsites probably form the basis of the observed differences between GH8 xylanases. GH8 xylanases, therefore, are an interesting group of enzymes, with potential towards engineering and applications.

  18. Investigation of 1D Siliver Nanoparticle Arrays for Use as Molecule Concentration-Specific SERS Substrates

    Directory of Open Access Journals (Sweden)

    Jun Gao


    Full Text Available One-dimensional arrays of silver nanoparticles with a particle size of 10.5 nm and interparticle spacing of 8.5 nm were fabricated by depositing nanoparticles in gas phase on block copolymer self-assembled templates. The substrate showed a surface-enhanced Raman scattering (SERS enhancement factor as high as 1×107 with good reproducibility and stability. The dependence of the average enhancement factor and the SERS intensity on the packing density of the analyte molecules were investigated. For a tiny amount of analytes in the range of 1×10-14 to 3×10-13 mol/mm2, the SERS signal showed a linear dependence on the molecule packing density on a logarithmic scale, with a slope of about 1.25. The substrates are promising for quantitative detection of trace level molecules.

  19. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity. (United States)

    Altmann, F; Schweiszer, S; Weber, C


    The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate.

  20. Alternative effector-function profiling identifies broad HIV-specific T-cell responses in highly HIV-exposed individuals who remain uninfected. (United States)

    Ruiz-Riol, Marta; Llano, Anuska; Ibarrondo, Javier; Zamarreño, Jennifer; Yusim, Karina; Bach, Vanessa; Mothe, Beatriz; Perez-Alvarez, Susana; Fernandez, Marco A; Requena, Gerard; Meulbroek, Michael; Pujol, Ferran; Leon, Agathe; Cobarsi, Patricia; Korber, Bette T; Clotet, Bonaventura; Ganoza, Carmela; Sanchez, Jorge; Coll, Josep; Brander, Christian


    The characterization of host immune responses to human immunodeficiency virus (HIV) in HIV controllers and individuals with high exposure but seronegativity to HIV (HESN) is needed to guide the development of effective preventive and therapeutic vaccine candidates. However, several technical hurdles severely limit the definition of an effective virus-specific T-cell response. By using a toggle-peptide approach, which takes HIV sequence diversity into account, and a novel, boosted cytokine staining/flow cytometry strategy, we here describe new patterns of T-cell responses to HIV that would be missed by standard assays. Importantly, this approach also allows detection of broad and strong virus-specific T-cell responses in HESN individuals that are characterized by a T-helper type 1 cytokine-like effector profile and produce cytokines that have been associated with potential control of HIV infection, including interleukin 10, interleukin 13, and interleukin 22. These results establish a novel approach to improve the current understanding of HIV-specific T-cell immunity and identify cellular immune responses and individual cytokines as potential markers of relative HIV resistance. As such, the findings also help develop similar strategies for more-comprehensive assessments of host immune responses to other human infections and immune-mediated disorders.

  1. General and Specific Effects on Cattell-Horn-Carroll Broad Ability Composites: Analysis of the Woodcock-Johnson III Normative Update Cattell-Horn-Carroll Factor Clusters across Development (United States)

    Floyd, Randy G.; McGrew, Kevin S.; Barry, Amberly; Rafael, Fawziya; Rogers, Joshua


    Many school psychologists focus their interpretation on composite scores from intelligence test batteries designed to measure the broad abilities from the Cattell-Horn-Carroll theory. The purpose of this study was to investigate the general factor loadings and specificity of the broad ability composite scores from one such intelligence test…

  2. Phosphorylation-Dependent PIH1D1 Interactions Define Substrate Specificity of the R2TP Cochaperone Complex

    Directory of Open Access Journals (Sweden)

    Zuzana Hořejší


    Full Text Available The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs, RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

  3. Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI. (United States)

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K


    BphI, a pyruvate-specific class II aldolase found in the polychlorinated biphenyls (PCBs) degradation pathway, catalyzes the reversible C-C bond cleavage of (4S)-hydroxy-2-oxoacids to form pyruvate and an aldehyde. Mutations were introduced into bphI to probe the contribution of active site residues to substrate recognition and catalysis. In contrast to the wild-type enzyme that has similar specificities for acetaldehyde and propionaldehyde, the L87A variant exhibited a 40-fold preference for propionaldehyde over acetaldehyde. The specificity constant of the L89A variant in the aldol addition reaction using pentaldehyde is increased ∼50-fold, making it more catalytically efficient for pentaldehyde utilization compared to the wild-type utilization of the natural substrate, acetaldehyde. Replacement of Tyr-290 with phenylalanine or serine resulted in a loss of stereochemical control as the variants were able to utilize substrates with both R and S configurations at C4 with similar kinetic parameters. Aldol cleavage and pyruvate α-proton exchange activity were undetectable in the R16A variant, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate. The pH dependence of the enzyme is consistent with a single deprotonation by a catalytic base with pK(a) values of approximately 7. In H20A and H20S variants, pH profiles show the dependence of enzyme activity on hydroxide concentration. On the basis of these results, a catalytic mechanism is proposed.

  4. Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters. (United States)

    Silverstein, Nechama; Ewers, David; Forrest, Lucy R; Fahlke, Christoph; Kanner, Baruch I


    Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

  5. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme (United States)

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P.-Y.; Wang, Steven S.-S.


    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer’s disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12–16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12–16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12–16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1–7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1–7)C and qf-Aβ(12–16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells. PMID:27096746

  6. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity. (United States)

    Todorov, Katherine Abold; Garcia, George A


    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  7. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme. (United States)

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P-Y; Wang, Steven S-S


    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

  8. The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi [Marquette Univ., Milwaukee, WI (United States); Maurice, Martin [Marquette Univ., Milwaukee, WI (United States)


    Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2. AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH fromGranulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr299 and Arg307, are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr299 and Arg307 were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr299 and Arg307 serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172. The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.

  9. The structure and substrate specificity of human Cdk12/Cyclin K (United States)

    Bösken, Christian A.; Farnung, Lucas; Hintermair, Corinna; Merzel Schachter, Miriam; Vogel-Bachmayr, Karin; Blazek, Dalibor; Anand, Kanchan; Fisher, Robert P.; Eick, Dirk; Geyer, Matthias


    Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases. PMID:24662513

  10. Substrate specificity of mammalian N-terminal α-amino methyltransferase (United States)

    Petkowski, Janusz J.; Schaner Tooley, Christine E.; Anderson, Lissa C.; Shumilin, Igor A.; Balsbaugh, Jeremy L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Minor, Wladek; Macara, Ian G.


    N-terminal methylation of free α-amino-groups is a post-translational modification of proteins that has been known for 30 years but has been very little studied. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono- di- or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-aminomethylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification, and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products. PMID:22769851

  11. An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles. (United States)

    Yeom, Soo-Jin; Kim, Hye-Jung; Lee, Jung-Kul; Kim, Dong-Eun; Oh, Deok-Kun


    Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated pi-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity.

  12. Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

    Directory of Open Access Journals (Sweden)


    Full Text Available Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.

  13. High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles. (United States)

    Figueroa-Teran, Rubi; Pak, Heidi; Blomquist, Gary J; Tittiger, Claus


    Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

  14. Matching the Diversity of Sulfated Biomolecules: Creation of a Classification Database for Sulfatases Reflecting Their Substrate Specificity (United States)

    Barbeyron, Tristan; Brillet-Guéguen, Loraine; Carré, Wilfrid; Carrière, Cathelène; Caron, Christophe; Czjzek, Mirjam; Hoebeke, Mark; Michel, Gurvan


    Sulfatases cleave sulfate groups from various molecules and constitute a biologically and industrially important group of enzymes. However, the number of sulfatases whose substrate has been characterized is limited in comparison to the huge diversity of sulfated compounds, yielding functional annotations of sulfatases particularly prone to flaws and misinterpretations. In the context of the explosion of genomic data, a classification system allowing a better prediction of substrate specificity and for setting the limit of functional annotations is urgently needed for sulfatases. Here, after an overview on the diversity of sulfated compounds and on the known sulfatases, we propose a classification database, SulfAtlas (, based on sequence homology and composed of four families of sulfatases. The formylglycine-dependent sulfatases, which constitute the largest family, are also divided by phylogenetic approach into 73 subfamilies, each subfamily corresponding to either a known specificity or to an uncharacterized substrate. SulfAtlas summarizes information about the different families of sulfatases. Within a family a web page displays the list of its subfamilies (when they exist) and the list of EC numbers. The family or subfamily page shows some descriptors and a table with all the UniProt accession numbers linked to the databases UniProt, ExplorEnz, and PDB. PMID:27749924

  15. Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337-->Val) in fructose intolerance. (United States)

    Rellos, P; Ali, M; Vidailhet, M; Sygusch, J; Cox, T M


    A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala337-->Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149-->Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 microM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 microM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant

  16. Rv2607 from Mycobacterium tuberculosis is a pyridoxine 5'-phosphate oxidase with unusual substrate specificity.

    Directory of Open Access Journals (Sweden)

    Ellene H Mashalidis

    Full Text Available Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx, an enzyme that oxidizes pyridoxine 5'-phosphate (PNP or pyridoxamine 5'-phosphate (PMP to form pyridoxal 5'-phosphate (PLP. We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat and K(M for this reaction were 0.01 s(-1 and 360 µM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.

  17. A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis. (United States)

    Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G


    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

  18. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.


    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  19. Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants. (United States)

    Lopes, Adriana R; Sato, Paloma M; Terra, Walter R


    Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Four insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera, and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization using different colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe p-nitroanilide). Chloromethyl ketones (TPCK, N- alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N- carbobenzoxy-Gly-Gly-Phe-CK) inactivated all chymotrypsins tested. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1) s(-1); Z-GGF-CK, 150 to 450 M(-1) s(-1)) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1) s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa value. This is proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed to be an adaptation to the presence of dietary ketones.

  20. Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase. (United States)

    Chow, Dar-Chone; Rice, Kacie; Huang, Wanzhi; Atmar, Robert L; Palzkill, Timothy


    The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (Ki = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (Ki = 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 β-lactamase. A genetic screen for BLIP function in Escherichia coli was used to narrow the binding specificity of BLIP by identifying amino acid substitutions that retain affinity for KPC-2 while losing affinity for TEM-1 β-lactamase. The combination of single substitutions yielded the K74T:W112D BLIP variant, which was shown by inhibition assays to retain high affinity for KPC-2 with a Ki of 0.4 nM, while drastically losing affinity for TEM-1 with a Ki > 10 μM. The K74T:W112D mutant therefore binds KPC-2 β-lactamase 3 times more tightly while binding TEM-1 > 20000-fold more weakly than wild-type BLIP. The K74T:W112D BLIP variant also exhibited low affinity (Ki > 10 μM) for other class A β-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 β-lactamase suggest it could be a useful sensor for the presence of this enzyme in multidrug-resistant bacteria. This was demonstrated with an assay employing BLIP K74T:W112D conjugated to a bead to specifically pull-down and detect KPC-2 β-lactamase in lysates from clinical bacterial isolates containing multiple β-lactamases.

  1. nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala. (United States)

    Lewin, A; Cervantes, E; Chee-Hoong, W; Broughton, W J


    Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.

  2. Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex* (United States)

    Cai, Yong; Jin, Jingji; Swanson, Selene K.; Cole, Michael D.; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P.; Conaway, Joan W.; Conaway, Ronald C.


    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8. PMID:20018852

  3. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Huiqing; Li, Xiaojing [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Zhao, Yuancong, E-mail: [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Li, Jingan; Chen, Jiang [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Yang, Ping, E-mail: [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Maitz, Manfred F. [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Max Bergmann Center of Biomaterials Dresden, Leibniz of Polymer Research Dresden, 01069 Dresden (Germany); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)


    Graphical abstract: The phospholipid groups of PMMDP can inhibit platele adhesion, and the EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. The catechol groups of PMMDP play a critical role as molecular anchor for balancing the binding between the coating and the substrate. - Highlights: • The uniform coating of PMMDP can be constructed on titanium surface successfully through the catechol groups. • The phospholipid groups of PMMDP can inhibit platele adhesion, fibrinogen denaturation and improve the hydrophilicity of substrate. • The EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. - Abstract: A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

  4. The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization. (United States)

    Ageitos, Jose Manuel; Yazawa, Kenjiro; Tateishi, Ayaka; Tsuchiya, Kousuke; Numata, Keiji


    The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.

  5. Elucidation of substrate specificity in Aspergillus nidulans UDP-galactose-4-epimerase.

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    Sean A Dalrymple

    Full Text Available The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD(+ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s(-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.

  6. Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear

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    Vianna D.M.L.


    Full Text Available The periaqueductal gray (PAG has been traditionally considered to be an exit relay for defensive responses. Functional mapping of its subdivisions has advanced our knowledge of this structure, but synthesis remains difficult mainly because results from lesion and stimulation studies have not correlated perfectly. After using a strategy that combined both techniques and a reevaluation of the available literature on PAG function and connections, we propose here that freezing could be mediated by different PAG subdivisions depending on the presence of immediate danger or exposure to related signaling cues. These subdivisions are separate functional entities with distinct descending and ascending connections that are likely to play a role in different defensive responses. The existence of ascending connections also suggests that the PAG is not simply a final common path for defensive responses. For example, the possibility that indirect ascending connections to the cingulate cortex could play a role in the expression of freezing evoked by activation of the neural substrate of fear in the dorsal PAG has been considered.

  7. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology


    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  8. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

    DEFF Research Database (Denmark)

    Ntokou, Eleni; Hansen, Lykke Haastrup; Kongsted, Jacob;


    homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X......Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence......-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore...

  9. Animal deoxyribonucleoside kinases: 'forward' and 'retrograde' evolution of their substrate specificity

    DEFF Research Database (Denmark)

    Piskur, Jure; Sandrini, Michael; Knecht, Wolfgang;


    specificity. Recent studies suggest that in the animal lineage one of the brogenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC, which subsequently, through remodelling...

  10. Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, M.D.; Garcia, M.C.; Sprecher, H. (Eastern Virginia Medical School, Norfolk (USA))


    Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14- tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-(14C)eicosatrienoate was two-to threefold that of 5,11,14-(14C)eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.

  11. Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.


    Besse, I.; Wong, J.H.; Kobrehel, K.; Buchanan, B.B.


    We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by...

  12. Lipase specificity towards eicosapentaenoic acid and docosahexaenoic acid depends on substrate structure. (United States)

    Lyberg, Ann-Marie; Adlercreutz, Patrick


    The fatty acid specificity of five lipases towards eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was evaluated in the hydrolysis of fish oil, squid oil and a model system. The model system contained methyl esters of EPA, DHA and palmitic acid. All the investigated lipases discriminated against both EPA and DHA more in the model system than in the natural oils. Thus both EPA and DHA were more easily hydrolysed from a glyceride than from a methyl ester. In the model system, the lipase from Candida rugosa showed the highest discrimination against DHA, while the lipases from Pseudomonas fluorescens and Pseudomonas cepacia discriminated against EPA the most. In a glyceride, the fatty acid specificity of lipases towards EPA and DHA was affected by the positional distribution of the fatty acids and the glyceride structure due to the regiospecificity and triglyceride specificity of the lipase. In the oils, the Pseudomonas lipases also discriminated against EPA the most, while DHA was initially discriminated the most by the lipase from Thermomyces lanuginosus. However, after longer reaction times the enrichment of DHA in the glyceride fraction of the oils was greatest for the lipase from C. rugosa.

  13. Substrate specificities of glycosidases from Aspergillus species pectinase preparations on elderberry anthocyanins. (United States)

    Pricelius, Sina; Murkovic, Michael; Souter, Philip; Guebitz, Georg M


    Attractive color is one of the most important sensory characteristics of fruit and berry products, and elderberry juice is widely used as natural colorant. When pectinase preparations were used in the production of elderberry juice for clarification, a concomitant decrease of anthocyanins and thus a color loss were observed. This paper demonstrates that this is due to side glycosidase activities contained in commercial pectinase preparations from Aspergillus sp. Using LC-MS, sequential deglycosylation of cyanidin-3-sambubioside, cy-3-glucoside, cy-3-sambubioside-5-glucoside, and cy-3,5-diglucoside was found to be catalyzed by specific glycosidases contained in the pectinase preparations. There was no big difference in the deglycosylation rate between monoglucosidic or diglucosidic anthocyanins. However, the degradation rate was decreased when rutinose was attached to cyanidin, whereas the structure of the aglycone itself had almost no influence. Pure beta-glucosidases from Agrobacterium species and Aspergillus niger and the beta-glucosidase N188 from A. niger did not show any conversion of anthocyanins, indicating the presence of specific glycosidases. Thus, an activity gel based assay was developed to detect anthocyanin-specific glycosidase activity in enzyme preparations, and according to LC-MS peptide mass mapping of digested bands, homologies to a beta-glucosidase from Aspergillus kawachii were found.

  14. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment. (United States)

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A


    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent

  15. Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity. (United States)

    Arndt, Joseph W; Chai, Qing; Christian, Todd; Stevens, Raymond C


    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.

  16. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions. (United States)

    Long, Yicheng; Jackman, Jane E


    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

  17. Excimer laser texturing of natural composite polymer surfaces for studying cell-to-substrate specific response (United States)

    Dinca, V.; Alloncle, P.; Delaporte, P.; Ion, V.; Rusen, L.; Filipescu, M.; Mustaciosu, C.; Luculescu, C.; Dinescu, M.


    Surface modifications of biocompatible materials are among the main factors used for enhancing and promoting specific cellular activities (e.g. spreading, adhesion, migration, and differentiation) for various types of medical applications such as implants, microfluidic devices, or tissue engineering scaffolds. In this work an excimer laser at 193 nm was used to fabricate chitosan-collagen roughness gradients. The roughness gradients were obtained in one step by applying single laser pulses and sample tilting. Fourier transform infrared spectroscopy measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and spectro-ellipsometry (SE) were used for sample characterization. The goal is to determine the optimal morpho-chemical characteristics of these structures for in vitro tailoring of protein adsorption and cell behavior. The response induced by the roughness gradient onto various cell lines (i.e. L 929 fibroblasts, HEP G2 hepatocytes, OLN 93 oligodendrocytes, M63 osteoblasts) and bovine serum albumin (BSA) protein absorption was investigated.

  18. Excimer laser texturing of natural composite polymer surfaces for studying cell-to-substrate specific response

    Energy Technology Data Exchange (ETDEWEB)

    Dinca, V., E-mail: [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Bucharest (Romania); Alloncle, P.; Delaporte, P. [Aix-Marseille University, CNRS, LP3 Laboratory, Campus de Luminy, 13288 Marseille (France); Ion, V. [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Bucharest (Romania); Faculty of Physics, University of Bucharest, 077125 Magurele (Romania); Rusen, L.; Filipescu, M. [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering – IFIN HH, Magurele, Bucharest (Romania); Luculescu, C.; Dinescu, M. [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Bucharest (Romania)


    Highlights: • Roughness gradients are obtained in one step by applying single laser pulses and sample tilting. • BSA protein and cell dependence behavior onto gradient characteristics was studied. • The degradation of the samples by lysozyme was correlated to its ability to access the textured area. - Abstract: Surface modifications of biocompatible materials are among the main factors used for enhancing and promoting specific cellular activities (e.g. spreading, adhesion, migration, and differentiation) for various types of medical applications such as implants, microfluidic devices, or tissue engineering scaffolds. In this work an excimer laser at 193 nm was used to fabricate chitosan–collagen roughness gradients. The roughness gradients were obtained in one step by applying single laser pulses and sample tilting. Fourier transform infrared spectroscopy measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and spectro-ellipsometry (SE) were used for sample characterization. The goal is to determine the optimal morpho-chemical characteristics of these structures for in vitro tailoring of protein adsorption and cell behavior. The response induced by the roughness gradient onto various cell lines (i.e. L 929 fibroblasts, HEP G2 hepatocytes, OLN 93 oligodendrocytes, M63 osteoblasts) and bovine serum albumin (BSA) protein absorption was investigated.

  19. Neural substrates of defensive reactivity in two subtypes of specific phobia. (United States)

    Lueken, Ulrike; Hilbert, Kevin; Stolyar, Veronika; Maslowski, Nina I; Beesdo-Baum, Katja; Wittchen, Hans-Ulrich


    Depending on threat proximity, different defensive behaviours are mediated by a descending neural network involving forebrain (distal threat) vs midbrain areas (proximal threat). Compared to healthy subjects, it can be assumed that phobics are characterized by shortened defensive distances on a behavioural and neural level. This study aimed at characterizing defensive reactivity in two subtypes of specific phobia [snake (SP) and dental phobics (DP)]. Using functional magnetic resonance imaging (fMRI), n = 39 subjects (13 healthy controls, HC; 13 SP; 13 DP) underwent an event-related fMRI task employing an anticipation (5-10 s) and immediate perception phase (phobic pictures and matched neutral stimuli; 1250 ms) to modulate defensive distance. Although no differential brain activity in any comparisons was observed in DP, areas associated with defensive behaviours (e.g. amygdala, hippocampus, midbrain) were activated in SP. Decreasing defensive distance in SP was characterized by a shift to midbrain activity. Present findings substantiate differences between phobia types in their physiological and neural organization that can be expanded to early stages of defensive behaviours. Findings may contribute to a better understanding of the dynamic organization of defensive reactivity in different types of phobic fear.

  20. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III. (United States)

    Richard-Fogal, Cynthia L; San Francisco, Brian; Frawley, Elaine R; Kranz, Robert G


    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  1. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    NARCIS (Netherlands)

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van


    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(meth

  2. Vascular peroxidase 1 catalyzes the formation of hypohalous acids: characterization of its substrate specificity and enzymatic properties. (United States)

    Li, Hong; Cao, Zehong; Zhang, Guogang; Thannickal, Victor J; Cheng, Guangjie


    The heme-containing peroxidase family comprises eight members in humans. The physiological and pathophysiological roles of heme-containing peroxidases are not well understood. Phagocyte-derived myeloperoxidase (MPO) utilizes chloride and bromide, in the presence of hydrogen peroxide (H(2)O(2)), to generate hypochlorous acid and hypobromous acid, potent oxidizing species that are known to kill invading pathogens. Vascular peroxidase 1 (VPO1) is a new member of the heme-containing peroxidase family; VPO1 is highly expressed in the cardiovascular system, lung, liver, pancreas, and spleen. However, functional roles of VPO1 have not been defined. In this report, we demonstrate the capacity for VPO1 to catalyze the formation of hypohalous acids, and characterize its enzymatic properties. VPO1, like MPO but unlike lactoperoxidase, is able to generate hypochlorous acid, hypobromous acid, and hypothiocyanous acid in the presence of H(2)O(2). Under physiological pH and concentrations of halides (100μM KBr, 100μM KSCN, and 100mM NaCl), VPO1 utilizes approximately 45% of H(2)O(2) for the generation of hypobromous acid, 35% for hypothiocyanous acid, and 18% for hypochlorous acid. The specific activity of VPO1 is ∼10- to 70-fold lower than that of MPO, depending on the specific substrate. These studies demonstrate that the enzymatic properties and substrate specificity of VPO1 are similar to MPO; however, significantly lower catalytic rate constants of VPO1 relative to MPO suggest the possibility of other physiologic roles for this novel heme-containing peroxidase.

  3. Plasmodium falciparum Plasmepsin V (PfPMV): Insights into recombinant expression, substrate specificity and active site structure. (United States)

    Boonyalai, Nonlawat; Sittikul, Pichamon; Yuvaniyama, Jirundon


    Plasmepsin V from Plasmodium falciparum (PfPMV) is responsible for the cleavage of the Plasmodium export element (PEXEL) motif at the N-terminus of several hundreds of the exported proteins. PfPMV is necessary for parasite viability and has become a novel promising target for antimalarial therapy. The first recombinant expression of soluble, active PfPMV as thioredoxin fusion proteins is reported herein. Two truncated forms of PfPMV were fused to thioredoxin (Trx) to generate Trx-PfPMVp37 and Trx-PfPMVm84. The fusion proteins were successfully purified using Ni(2+) affinity chromatography in combination with ATP treatment to eliminate Escherichia coli HSP60 contaminant. Trx-PfPMVm84 could hydrolyze the PEXEL-containing peptides more efficiently than Trx-PfPMVp37. Interestingly, both Trx-PfPMVs preferred to cleave PfEMP2 peptide over HRPII peptide. The replacement of Ser with Val or Glu at P1' position created a substrate with 75% reduction in the enzyme activity, whereas the substitution of Ile with Lys or Glu at P2 position reduced the cleavage efficiency by 30%. The activity of Trx-PfPMVm84 was inhibited by PMSF and nelfinavir but not by pepstatin A. After the removal of Trx domain, activities of both enzymes toward PfEMP2 and HRPII peptides were fitted to the Michaelis-Menten model to determine kinetic parameters. The Km values toward both peptides were apparently much lower than the previously reported data although with similar kcat values. Along with an improved PfPMV preparation protocol, these findings have provided insights into its substrate specificity at P2 and P1' positions as well as interactions among the enzyme, substrates, and inhibitors.

  4. Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.

    Directory of Open Access Journals (Sweden)

    Lisandro J González


    Full Text Available Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs, enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains "atypical" second sphere residues (S84, G121. Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.

  5. Milk secretion of nitrofurantoin, as a specific BCRP/ABCG2 substrate, in assaf sheep: modulation by isoflavones. (United States)

    Pérez, M; Real, R; Mendoza, G; Merino, G; Prieto, J G; Alvarez, A I


    Studies on residues in milk used for human consumption have increased due to health concerns and priority interest in the control of potentially risky drugs. The protein BCRP/ABCG2, present in the mammary epithelia, actively extrudes drugs into milk and can be modulated by isoflavones. Nitrofurantoin is a specific BCRP substrate which is actively excreted into milk by this transporter. In this research, we studied nitrofurantoin transport into milk in four experimental groups: G1-calves fed forage with isoflavones; G2-calves fed forage with isoflavones and administered exogenous genistein and daidzein; G3-calves fed forage without isoflavones; G4-calves fed forage without isoflavones and administered exogenous genistein and daidzein. Results show increased levels of nitrofurantoin in milk from calves without isoflavones (G3) and decreased nitrofurantoin residues in milk when isoflavones were present, either by forage (G1 and G2) or by exogenous administration (G4). The values of C(max) in milk were significantly lower in those groups with isoflavones in forage (G1, G2). Plasma levels were low and unmodified among the groups. Inter-individual variation was high. All these results seem to point to a feasible control of drug secretion into milk through isoflavones in the diet when the drug is a good BCRP/ABCG2 substrate.

  6. Identification of ω-aminotransferase from Caulobacter crescentus and site-directed mutagenesis to broaden substrate specificity. (United States)

    Hwang, Bum-Yeol; Ko, Seung-Hyun; Park, Hyung-Yeon; Seo, Joo-Hyun; Lee, Bon-Su; Kim, Byung-Gee


    A putative aminotransferase gene, cc3143 (aptA), from Caulobacter crescentus was screened by bioinformatical tools and overexpressed in E. coli, and the substrate specificity of the aminotransferase was investigated. AptA showed high activity for short-chain beta-amino acids. It showed the highest activity for 3-amino-n-butyric acid. It showed higher activity toward aromatic amines than aliphatic amines. The 3D model of the aminotransferase was constructed by homology modeling using a dialkylglycine decarboxylase PDB ID: 1DGE) as a template. Then, the aminotransferase was rationally redesigned to increase the activity for 3-amino- 3-phenylpropionic acid. The mutants N285A and V227G increased the relative activity for 3-amino-3-phenylpropionic acid to 3-amino-n-butyric acid by 11-fold and 3-fold, respectively, over that of wild type.

  7. Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase. (United States)

    Miklaszewska, Magdalena; Banaś, Antoni


    Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols.

  8. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    Directory of Open Access Journals (Sweden)

    Silvija Miosic

    Full Text Available During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin and two (cyanidin hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring, whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  9. Substrate Specificity and Ionic Regulation of GlnPQ from Lactococcus lactis. An ATP-Binding Cassette Transporter with Four Extracytoplasmic Substrate-Binding Domains

    NARCIS (Netherlands)

    Schuurman-Wolters, Gea K.; Poolman, Bert


    We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmi

  10. Phosphoramidate pronucleotides: a comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins. (United States)

    Chou, Tsui-Fen; Baraniak, Janina; Kaczmarek, Renata; Zhou, Xin; Cheng, Jilin; Ghosh, Brahma; Wagner, Carston R


    To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety of pronucleotide approaches have been developed. Our laboratory and others have demonstrated that nucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphate nucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for their bioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bonds between nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability of nucleosides to quench the fluorescence of covalently linked amines containing indole, a sensitive, continuous fluorescence-based assay was developed. A series of substrates linking the naturally fluorogenic indole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kinetic parameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize the elemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosine phosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificity constant (kcat/Km) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preference was not observed for E. coli hinT, hHint1 exhibited a 300-fold preference for d-tryptophan phosphoramidates over l-isomers. The most efficient substrates evaluated to date are those that contain the less sterically hindering amine leaving group, tryptamine, with kcat and Km values comparable to those found for adenosine kinase. The apparent second-order rate constants (kcat/Km) for adenosine tryptamine phosphoramidate monoester were found to be 107 M-1 s-1 for hHint1 and 106 M-1 s-1 for E. coli hinT. Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observed hydrogen bonding between the 2'-OH group of adenosine monophosphate and the

  11. Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. (United States)

    Stengl, Bernhard; Reuter, Klaus; Klebe, Gerhard


    Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.

  12. Specific amino acid residues are involved in substrate discrimination and template binding of human REV1 protein. (United States)

    Piao, Jinlian; Masuda, Yuji; Kamiya, Kenji


    REV1 is a member of the Y-family DNA polymerases, but is atypical in utilizing only dCTP with a preference for guanine (G) as the template. Crystallography of the REV1-DNA-dCTP ternary complex has revealed a unique mechanism by which template G is evicted from the DNA helix and incoming dCTP is recognized by an arginine residue in an alpha-loop, termed the N-digit. To better understand functions of its individual amino acid residues, we made a series of mutant human REV1 proteins. We found that R357 and L358 play vital roles in template binding. Furthermore, extensive mutation analysis revealed a novel function of R357 for substrate discrimination, in addition to previously proposed specific interaction with incoming dCTP. We found that the binding pocket for dCTP of REV1 has also significant but latent affinity for dGTP. The results suggest that the positive charge on R357 could prevent interaction with dGTP. We propose that both direct and indirect mechanisms mediated by R357 ensure specificity for dCTP.

  13. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling (United States)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.


    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  14. Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1* (United States)

    Paumann-Page, Martina; Katz, Romy-Sophie; Bellei, Marzia; Schwartz, Irene; Edenhofer, Eva; Sevcnikar, Benjamin; Soudi, Monika


    Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV. PMID:28154175

  15. Antimicrobial activity of cathelicidins BMAP28, SMAP28, SMAP29, and PMAP23 against Pasteurella multocida is more broad-spectrum than host species specific. (United States)

    Brogden, Kim A; Nordholm, Gwen; Ackermann, Mark


    The antimicrobial activity of linear, cationic alpha-helical peptides from cattle (BMAP28), sheep (SMAP28 and SMAP29), and pigs (PMAP23) were assessed to determine if activity was selective for Pasteurella multocida from a particular animal species or broad-spectrum against all P. multocida tested. The antimicrobial activities of synthetic peptides were determined for P. multocida isolated from cattle (10 isolates), sheep (10 isolates), and pigs (10 isolates) in a broth microdilution assay. All thirty isolates of P. multocida were susceptible to BMAP28 (MICs and MBCs, 1.0-1.9 microM); SMAP28 and SMAP29 (MICs and MBCs, 0.2-0.7 microM); and PMAP23 (MICs and MBCs, 4.3 to > or = 6.8 microM). Overall, the results of this study suggest that synthesized cathelicidins from cattle, sheep, and pigs had broad-spectrum antimicrobial activity against all P. multocida.

  16. Broad-based nutritional supplementation in 3xTg mice corrects mitochondrial function and indicates sex-specificity in response to Alzheimer's disease intervention. (United States)

    Wolf, Andrew B; Braden, B Blair; Bimonte-Nelson, Heather; Kusne, Yael; Young, Nicole; Engler-Chiurazzi, Elizabeth; Garcia, Alexandra N; Walker, Douglas G; Moses, Guna S D; Tran, Hung; LaFerla, Frank; Lue, LihFen; Emerson Lombardo, Nancy; Valla, Jon


    Nutrition has been highlighted as a potential factor in Alzheimer's disease (AD) risk and decline and has been investigated as a therapeutic target. Broad-based combination diet therapies have the potential to simultaneously effect numerous protective and corrective processes, both directly (e.g., neuroprotection) and indirectly (e.g., improved vascular health). Here we administered either normal mouse chow with a broad-based nutritional supplement or mouse chow alone to aged male and female 3xTg mice and wildtype (WT) controls. After approximately 4 months of feeding, mice were given a battery of cognitive tasks and then injected with a radiolabeled glucose analog. Brains were assessed for differences in regional glucose uptake and mitochondrial cytochrome oxidase activity, AD pathology, and inflammatory markers. Supplementation induced behavioral changes in the 3xTg, but not WT, mice, and the mode of these changes was influenced by sex. Subsequent analyses indicated that differential response to supplementation by male and female 3xTg mice highlighted brain regional strategies for the preservation of function. Several regions involved have been shown to mediate responses to steroid hormones, indicating a mechanism for sex-based vulnerability. Thus, these findings may have broad implications for the human response to future therapeutics.

  17. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP. (United States)

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders


    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  18. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates (United States)

    Chen, Huiqing; Li, Xiaojing; Zhao, Yuancong; Li, Jingan; Chen, Jiang; Yang, Ping; Maitz, Manfred F.; Huang, Nan


    A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

  19. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate. (United States)

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred


    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

  20. Epb41l5 competes with Delta as a substrate for Mib1 to coordinate specification and differentiation of neurons. (United States)

    Matsuda, Miho; Rand, Kinneret; Palardy, Greg; Shimizu, Nobuyuki; Ikeda, Hiromi; Dalle Nogare, Damian; Itoh, Motoyuki; Chitnis, Ajay B


    We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

  1. Molecular cloning, substrate specificity of the functionally expressed dihydroflavonol 4-reductases from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism. (United States)

    Fischer, Thilo C; Halbwirth, Heidrun; Meisel, Barbara; Stich, Karl; Forkmann, Gert


    Treatment with the dioxygenase inhibitor prohexadione-Ca leads to major changes in the flavonoid metabolism of apple (Malus domestica) and pear (Pyrus communis) leaves. Accumulation of unusual 3-deoxyflavonoids is observed, which have been linked to an enhanced resistance toward fire blight. The committed step in this pathway is the reduction of flavanones. Crude extracts from leaves are able to perform this reaction. There was previous evidence that DFR enzymes of certain plants possess additional flavanone 4-reductase (FNR) activity. Such an FNR activity of DFR enzymes is proved here by heterologous expression of the enzymes. The heterologously expressed DFR/FNR enzymes of Malus and Pyrus possess distinct differences in substrate specificities despite only minor differences of the amino acid sequences. Kinetic studies showed that dihydroflavonols generally are the preferred substrates. However, with the observed substrate specificities the occurrence of 3-deoxyflavonoids in vivo after application of prohexadione-Ca can be explained.

  2. DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications. (United States)

    Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da


    Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM ( is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

  3. Substrate specificity of the bovine serum amine oxidase and in situ characterisation of aminoaldehydes by NMR spectroscopy. (United States)

    Houen, Gunnar; Struve, Casper; Søndergaard, Roar; Friis, Tina; Anthoni, Uffe; Nielsen, Per H; Christophersen, Carsten; Petersen, Bent O; Duus, Jens Ø


    The oxidation of spermidine or homospermidine with bovine serum amine oxidase (BSAO) was monitored in situ, using proton nuclear magnetic resonance spectroscopy in water with 10% D(2)O. NMR assignments were performed by spin decoupling and COSY spectra or by comparison with data from synthetic aminoaldehydes. The results represent the first in situ characterisation of the highly reactive aminoaldehydes and showed oxidation at the N(1) amino group of spermidine and homospermidine. Comparison of homospermidine with a variety of substrates revealed that among straight chain di- and polyamines both an aminopropyl group and two primary amino groups separated by seven (norspermidine) or eight (spermidine) carbon atoms were required for optimal substrate ability. However, highest activity was seen with the substrate N-(4-aminobutyl)hexahydropyrimidine, showing that the substrate channel of BSAO has a dual substrate preference, with moderately bulky substituents at the distal end of a diamine contributing equally well as an alkyl amino group. Cytotoxic investigations of a variety of substrates for BSAO, confirmed previous results, that cytotoxicity is primarily linked to polyamines encompassing the aminopropyl moiety. No acrolein was observed at any time during the oxidation showing that it reacts very fast with available amino groups forming a variety of derivatives.

  4. New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Aduri, Nanda Gowtham; Jensen, Johanne Mørch


    Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of E. coli POTs YdgR, YhiP and YjdL were investigated by means of a la...

  5. Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays. (United States)

    Shirafuji, Hiroaki; Yazaki, Ryu; Shuto, Yozo; Yanase, Tohru; Kato, Tomoko; Ishikura, Youji; Sakaguchi, Zenjiro; Suzuki, Moemi; Yamakawa, Makoto


    TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.

  6. Molecular cloning and characterization of two YGL039w genes encoding broad specificity NADPH-dependent aldehyde reductases from Kluyveromyces marxianus strain DMB1. (United States)

    Akita, Hironaga; Watanabe, Masahiro; Suzuki, Toshihiro; Nakashima, Nobutaka; Hoshino, Tamotsu


    Two genes from Kluyveromyces marxianus strain DMB1, YGL039w1 and YGL039w2, encode putative uncharacterized oxidoreductases that respectively share 42 and 44% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase (EC To determine the enzymatic characteristics of their products, the two genes were expressed in recombinant Escherichia coli cells, after which the YGL039w1 and YGL039w2 proteins were purified to homogeneity. In the presence of NADPH, both enzymes showed reductive activities toward at least nine aldehyde substrates, but no NADP(+)-dependent oxidative activities. These two YGL039w proteins thus appear to be aldehyde reductases. In addition, although both enzymes retained more than 70% of their activities after incubation for 30 min at temperatures below 40°C or at pHs between 5.5 and 11.3, YGL039w2 was slightly more thermostable than YGL039w1.

  7. Comparison of a Broad-Based Screen versus Disorder-Specific Screen in Detecting Young Children with an Autism Spectrum Disorder (United States)

    Wiggins, Lisa D; Piazza, Vivian; Robins, Diana L


    The goals of our study were to (a) compare agreement between autism spectrum disorder diagnosis and outcome of the Modified Checklist for Autism in Toddlers and Parents Evaluation of Developmental Status in a sample of toddlers and (b) examine specific concerns noted for toddlers who screened negative on the Modified Checklist for Autism in…

  8. Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase. (United States)

    Astegno, Alessandra; Allegrini, Alessandra; Piccoli, Stefano; Giorgetti, Alejandro; Dominici, Paola


    In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

  9. Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice (United States)

    Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming


    Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction. PMID:26305669

  10. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations. (United States)

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A; Andri, Sergio; Savi, Tadeja; Nardini, Andrea


    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed.

  11. A Coherent Study of Emission Lines from Broad-Band Photometry: Specific Star-Formation Rates and [OIII]/H{\\beta} Ratio at 3 < z < 6

    CERN Document Server

    Faisst, A L; Hsieh, B C; Laigle, C; Salvato, M; Tasca, L; Cassata, P; Davidzon, I; Ilbert, O; Fevre, O Le; Masters, D; McCracken, H J; Steinhardt, C; Silverman, J D; De Barros, S; Hasinger, G; Scoville, N Z


    We measure the H{\\alpha} and [OIII] emission line properties as well as specific star-formation rates (sSFR) of spectroscopically confirmed 33 cannot be fully explained in a picture of cold accretion driven growth. We find a progressively increasing [OIII]{\\lambda}5007/H{\\beta} ratio out to z~6, consistent with the ratios in local galaxies selected by increasing H{\\alpha} EW (i.e., sSFR). This demonstrates the potential of using "local high-z analogs" to investigate the spectroscopic properties and relations of galaxies in the re-ionization epoch.

  12. pKa modulation of the acid/base catalyst within GH32 and GH68: a role in substrate/inhibitor specificity?

    Directory of Open Access Journals (Sweden)

    Shuguang Yuan

    Full Text Available Glycoside hydrolases of families 32 (GH32 and 68 (GH68 belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates and fructosyltransferases (sucrose/fructans as donor and acceptor substrates. In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif rather than the previously proposed Tyr motif (not conserved provides the proton to increase the pKa of the acid-base catalyst.

  13. Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

    Institute of Scientific and Technical Information of China (English)

    WU Hai-hua; ZHU Kun-yan; GUO Ya-ping; ZHANG Xiao-min; MA En-bo


    Specific activity, substrate specificity, and kinetic parameters (Km and Vmax) of glutathione S-transferases (GSTs) towards three substrates, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and p-nitrobenzene chloride (pNBC) were investigated in six tissues (foregut, midgut, hindgut, fat body, hemolymph, and muscle) of Oxya chinensis. In addition, the inhibition in vitro (ethacrynic acid, and Cibacron Blue 3GA) of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body > midgut > hindgut > muscle > foregut > hemolymph both in females and males. Glutathione 5-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0-fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration (IC50) values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.

  14. Structural characterization of mouse neutrophil serine proteases and identification of their substrate specificities: relevance to mouse models of human inflammatory diseases. (United States)

    Kalupov, Timofey; Brillard-Bourdet, Michèle; Dadé, Sébastien; Serrano, Hélène; Wartelle, Julien; Guyot, Nicolas; Juliano, Luiz; Moreau, Thierry; Belaaouaj, Azzaq; Gauthier, Francis


    It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases.

  15. Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis. (United States)

    Sorci, Leonardo; Cimadamore, Flavio; Scotti, Stefania; Petrelli, Riccardo; Cappellacci, Loredana; Franchetti, Palmarisa; Orsomando, Giuseppe; Magni, Giulio


    Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.

  16. Substrate specificity and recognition is conferred by the pleckstrin homology domain of the Dbl family guanine nucleotide exchange factor P-Rex2. (United States)

    Joseph, Raji E; Norris, F A


    Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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

  18. Energy and water use by invasive goats (Capra hircus) in an Australian rangeland, and a caution against using broad-scale allometry to predict species-specific requirements. (United States)

    Munn, A J; Cooper, C E; Russell, B; Dawson, T J; McLeod, S R; Maloney, S K


    Feral goats (Capra hircus) are ubiquitous across much of Australia's arid and semi-arid rangelands, where they compete with domestic stock, contribute to grazing pressure on fragile ecosystems, and have been implicated in the decline of several native marsupial herbivores. Understanding the success of feral goats in Australia may provide insights into management strategies for this and other invasive herbivores. It has been suggested that frugal use of energy and water contributes to the success of feral goats in Australia, but data on the energy and water use of free-ranging animals are lacking. We measured the field metabolic rate and water turnover rate of pregnant and non-pregnant feral goats in an Australian rangeland during late summer (dry season). Field metabolic rate of pregnant goats (601 ± 37 kJ kg(-0.73)d(-1)) was 1.3 times that of non-pregnant goats (456 ± 24 kJ kg(-0.73)d(-1)). The water turnover rate of pregnant goats (228 ± 18 mL kg(-0.79)d(-1)) was also 1.3 times that of non-pregnant goats (173 ± 18 kg(-0.79)d(-1)), but the difference was not significant (P=0.07). There was no significant difference in estimated dry matter digestibility between pregnant and non-pregnant goats (mean ca. 58%), blood or urine osmolality, or urine electrolyte concentrations, indicating they were probably eating similar diets and were able to maintain osmohomeostasis. Overall, the metabolic and hygric physiology of non-pregnant goats conformed statistically to the predictions for non-marine, non-reproductive placental mammals according to both conventional and phylogenetically independent analyses. That was despite the field metabolic rate and estimated dry matter intake of non-pregnant goats being only 60% of the predicted level. We suggest that general allometric analyses predict the range of adaptive possibilities for mammals, but that specific adaptations, as present in goats, result in ecologically significant departures from the average allometric curve. In

  19. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy. (United States)

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico


    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  20. X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases (United States)

    Prokofev, I. I.; Lashkov, A. A.; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.


    In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2'-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation

  1. Crystal Structure of the LasA Virulence Factor from Pseudomonas aeruginosa: Substrate Specificity and Mechanism of M23 Metallopeptidases

    Energy Technology Data Exchange (ETDEWEB)

    Spencer, James; Murphy, Loretta M.; Conners, Rebecca; Sessions, Richard B.; Gamblin, Steven J. (Wales); (Bristol Med Sci); (NIMR)


    Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.

  2. Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

    DEFF Research Database (Denmark)

    Söderhielm, Pella C; Andersen, Jacob; Munro, Lachlan


    of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram....... At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent...

  3. Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2O2 (United States)

    Cenni, Vittoria; Bavelloni, Alberto; Beretti, Francesca; Tagliavini, Francesca; Manzoli, Lucia; Lattanzi, Giovanna; Maraldi, Nadir M.; Cocco, Lucio; Marmiroli, Sandra


    Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization databases in combination with an anti–phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C2C12 myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions. PMID:21737686

  4. Structure and specificity of a new class of Ca(2+) independent housekeeping sortase from Streptomyces avermitilis provides insights into its non-canonical substrate preference. (United States)

    Das, Sreetama; Pawale, Vijaykumar S; Dadireddy, Venkatareddy; Singh, Avinash Kumar; Ramakumar, Suryanarayanarao; Roy, Rajendra P


    Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates Protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan Lipid II precursor. Sortase-mediated ligation ('sortagging') of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca(2+) and has been applied extensively for protein conjugations. Although majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca(2+) requirement (particularly for in cellulo applications) remains a drawback. Given that Gram-positive bacterial genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild type SaSrtA. Here we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca(2+)-independent activity and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca(2+) independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

  5. Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation. (United States)

    Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas


    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

  6. Altering substrate specificity of catechol 2,3-dioxygenase from Planococcus sp. strain S5 by random mutagenesis. (United States)

    Hupert-Kocurek, Katarzyna; Wojcieszyńska, Danuta; Guzik, Urszula


    c23o gene, encoding catechol 2,3-dioxygenase from Planococcus sp. strain S5 was randomly mutagenized to generate variant forms of the enzyme with higher degradation activity. Additionally, the effect of introduced mutations on the enzyme structure was analyzed based on the putative 3D models the wild-type and mutant enzymes. C23OB58 and C23OB81 mutant proteins with amino acid substitutions in close proximity to the enzyme surface or at the interface and in the vicinity of the enzyme active site respectively showed the lowest activity towards all catecholic substrates. The relative activity of C23OC61 mutant towards para-substituted catechols was 20-30% lower of the wild-type enzyme. In this mutant all changes: F191I, C268R, Y272H, V280A and Y293D were located within the conserved regions of C-terminal domain. From these F191I seems to have significant implications for enzyme activity. The highest activity towards different catechols was found for mutant C23OB65. R296Q mutation improved the activity of C23O especially against 4-chlorocatechol. The relative activity of above-mentioned mutant detected against this substrate was almost 6-fold higher than the wild-type enzyme. These results should facilitate future engineering of the enzyme for bioremediation.

  7. The 2010 Broad Prize (United States)

    Education Digest: Essential Readings Condensed for Quick Review, 2011


    A new data analysis, based on data collected as part of The Broad Prize process, provides insights into which large urban school districts in the United States are doing the best job of educating traditionally disadvantaged groups: African-American, Hispanics, and low-income students. Since 2002, The Eli and Edythe Broad Foundation has awarded The…

  8. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus. (United States)

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas


    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  9. Optimization of the fermentation conditions and substrate specifity of mycelium-bound ester hydrolases of Aspergillus oryzae Cs007

    Directory of Open Access Journals (Sweden)

    de Hong Yan


    Full Text Available In order to improve mycelium-bound ester hydrolases activities of Aspergillus oryzae Cs007, the main production conditions were investigated. The ester hydrolases activities were simultaneously determined by titration assay and spectrophotometric assay methods, using olive oil and p-nitrophenyl esters as substrates, respectively. The optimum carbon source and nitrogen source were olive oil and peptone, with the concentrations of 1% and 2.2%, respectively. The effects of carbon source, nitrogen source and their concentrations on the production of enzymes were identical when the enzymes activities were assayed by the two methods. The mycelium-bound enzymes showed hydrolytic activity toward all the tested p-nitrophenyl esters, triglycerides and fatty acid ethyl esters. But it showed greater preference for long-chain triglycerides and short-chain p-nitrophenyl esters.

  10. Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: altered substrate specificity and thermostability. (United States)

    Rajput, Rinky; Tiwary, Ekta; Sharma, Richa; Gupta, Rani


    Pro-sequences were swapped in cis between keratinases from Bacillus licheniformis (Ker BL) and Bacillus pumilus (Ker BP) to construct Ker ProBP-BL and Ker ProBL-BP, respectively. Expression of these keratinases was carried out constitutively by E. coli HB101-pEZZ18 system. They were characterized with respect to their parent enzymes, Ker BL and Ker BP, respectively. Ker ProBP-BL became more thermostable with a t(1/2) of 45 min at 80°C contrary to Ker BL which was not stable beyond 60°C. Similarly, the activity of Ker ProBP-BL on keratin and casein substrate, i.e. K:C ratio increased to 1.2 in comparison to 0.1 for Ker BL. Hydrolysis of insulin B-chain revealed that the cleavage sites increased to six from four in case of Ker ProBP-BL in comparison to Ker BL. However, cleavage sites decreased from seven to four in case of Ker ProBL-BP in comparison to the parent keratinase, Ker BP. Likewise, Ker ProBL-BP revealed altered pH and temperature kinetics with optima at pH 10 and 60°C in comparison to Ker BP which had optima at pH 9 and 70°C. It also cleaved soluble substrates with better efficiency in comparison to Ker BP with K:C ratio of 1.6. Pro-sequence mediated conformational changes were also observed in trans and were almost similar to the features acquired by the chimeras constructed in cis by swapping the pro-sequence region.

  11. The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues. (United States)

    Ouzzine, Mohamed; Gulberti, Sandrine; Levoin, Nicolas; Netter, Patrick; Magdalou, Jacques; Fournel-Gigleux, Sylvie


    The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His(308) by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate.

  12. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Directory of Open Access Journals (Sweden)

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  13. Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity. (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Kelly, Marcus P; Ramakrishna, Venky; Vitale, Laura; He, Li-Zhen; Keler, Tibor; Odunsi, Kunle; Old, Lloyd J; Ritter, Gerd; Gnjatic, Sacha


    Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

  14. Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applicatons in agriculture

    Energy Technology Data Exchange (ETDEWEB)

    Dirk, Lynnette M.A.; Schmidt, Jack J.; Cai, Yiying; Barnes, Jonathan C.; Hanger, Katherine M.; Nayak, Nihar R.; Williams, Mark A.; Grossman, Robert B.; Houtz, Robert L.; Rodgers, David W. (Kentucky)


    The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC], a plant specific deformylase, has been determined at a resolution of 2.4 {angstrom} (1 {angstrom}=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr{sup 178} as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr{sup 2} in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr{sup 178} to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr{sup 178} can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

  15. Vascular and hormonal responses to arginine: provision of substrate for nitric oxide or non-specific effect? (United States)

    MacAllister, R J; Calver, A L; Collier, J; Edwards, C M; Herreros, B; Nussey, S S; Vallance, P


    1. The vascular and hormonal effects of L- and D-arginine were compared in healthy subjects and in patients with insulin-dependent diabetes mellitus or untreated essential hypertension. 2. Infusion of L- or D-arginine (40 mumol/l) in the forearm vascular bed, sufficient to increase the local concentration approximately 20-fold, had no effect on blood flow or the vasodilator response to acetylcholine (30 and 100 nmol/min) in patients with insulin-dependent diabetes (n = 7) or essential hypertension (n = 7), or in age- and sex-matched control subjects (n = 7 in both groups). 3. Systemic infusion of 10 g of L-arginine (n = 5) or D-arginine (n = 3) increased plasma concentration of arginine approximately 20-fold without altering supine or erect haemodynamics. Increases in plasma insulin, prolactin and glucagon were seen with both enantiomers. The stereopurity of arginine was confirmed in a cell-culture assay system. 4. We conclude that, in healthy subjects and patients with essential hypertension or insulin-dependent diabetes, synthesis of nitric oxide within the vasculature is not limited by substrate availability. At high concentrations of arginine, non-stereospecific effects, including alterations in hormone concentration, occur. It remains to be determined whether these non-stereospecific hormonal changes might contribute to certain haemodynamic effects of arginine.

  16. Extension of Drosophila lifespan by cinnamon through a sex-specific dependence on the insulin receptor substrate chico. (United States)

    Schriner, Samuel E; Kuramada, Steven; Lopez, Terry E; Truong, Stephanie; Pham, Andrew; Jafari, Mahtab


    Cinnamon is a spice commonly used worldwide to flavor desserts, fruits, cereals, breads, and meats. Numerous health benefits have been attributed to its consumption, including the recent suggestion that it may decrease blood glucose levels in people with diabetes. Insulin signaling is an integral pathway regulating the lifespan of laboratory organisms, such as worms, flies, and mice. We posited that if cinnamon truly improved the clinical signs of diabetes in people that it would also act on insulin signaling in laboratory organisms and increase lifespan. We found that cinnamon did extend lifespan in the fruit fly, Drosophila melanogaster. However, it had no effect on the expression levels of the 3 aging-related Drosophila insulin-like peptides nor did it alter sugar, fat, or soluble protein levels, as would be predicted. In addition, cinnamon exhibited no protective effects in males against oxidative challenges. However, in females it did confer a protective effect against paraquat, but sensitized them to iron. Cinnamon provided no protective effect against desiccation and starvation in females, but sensitized males to both. Interestingly, cinnamon protected both sexes against cold, sensitized both to heat, and elevated HSP70 expression levels. We also found that cinnamon required the insulin receptor substrate to extend lifespan in males, but not females. We conclude that cinnamon does not extend lifespan by improving stress tolerance in general, though it does act, at least in part, through insulin signaling.


    Directory of Open Access Journals (Sweden)

    K. V. Avdiyuk


    Full Text Available The ability of Aspergillus flavus var. oryzae 80428 and Bacillus subtilis 147 α-amylases to split different carbohydrate-containing substrates, such as maltose, sucrose, trehalose, dextrin, α- and β-cyclodextrin, amylose, amylopectin, glycogen, pullulan, soluble starch, insoluble starch, corn starch, wheat starch, dextran 500 has been studied. It was shown that investigated enzymes differ by substrate specificity. α-Amylase of A. flavus var. oryzae 80428 rapidly hydrolysed soluble potato and wheat starch, while the α-amylase of B. subtilis 147 — only wheat starch. Both enzymes don’t cleave maltose, α-cyclodextrin and dextran 500. A. flavus var. oryzae 80428 α-amylase display very small ability to hydrolyze pullulan, while α-amylase of B. subtilis 147 it does not act in general. The lowest values of Michaelis constant for both enzymes at splitting of glycogen have been obtained, indicating that enzymes have the greatest affinity to this substrate. The studies of influence of chemically active substances on activity of A. flavus var. oryzae 80428 and B. subtilis 147 ?-amylases show there are resistant to urea, deoxycholic acid, Tween-80, Triton X-100 and hydrogen peroxide. It’s indicate the enzymes tested may be competitive in compare with earlier described in literature enzymes. The obtained results give a possibility to propose in future usage these enzymes in different fields of industry, foremost in detergent industry.

  18. FS laser processing of bio-polymer thin films for studying cell-to-substrate specific response (United States)

    Daskalova, A.; Nathala, Chandra S. R.; Kavatzikidou, P.; Ranella, A.; Szoszkiewicz, R.; Husinsky, W.; Fotakis, C.


    The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell's responses to substrate morphology. Mice fibroblasts migration was monitored after 3 days cultivation period using FESEM. We found that fibroblasts cells tend to migrate and adhere along the laser modified zones. The performed study proved that the immobilized collagen based biofilms suite as a template for successful fibroblasts cell guidance and orientation. Fs laser induced morphological modification of biomimetic materials exhibit direct control over fibroblasts behaviour due to induced change in their wettability state.

  19. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes* (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu


    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  20. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes. (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu


    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

  1. FS laser processing of bio-polymer thin films for studying cell-to-substrate specific response

    Energy Technology Data Exchange (ETDEWEB)

    Daskalova, A., E-mail: [Institute of Electronics, Bulgarian Academy of Sciences, 72, Tsarigradsko Chaussee Blvd., 1784 Sofia (Bulgaria); Nathala, Chandra S.R. [Institute of General Physics, Vienna University of Technology, Wiedner Hauptstr. 8-10/134, A-1040 Wien (Austria); Spectra-Physics Vienna, Fernkorngasse 10, 1100 Wien (Austria); Kavatzikidou, P.; Ranella, A. [Institute for Electronic Structure and Lasers-FORTH, P.O. Box 1385, Vassilika Vouton, 711 10 Heraklion, Crete (Greece); Szoszkiewicz, R. [Faculty of Materials Science and Engineering, Warsaw University of Technology, 141 Woloska Str., 02-507 Warsaw, Poland (Poland); Husinsky, W. [Institute of General Physics, Vienna University of Technology, Wiedner Hauptstr. 8-10/134, A-1040 Wien (Austria); Fotakis, C. [Institute for Electronic Structure and Lasers-FORTH, P.O. Box 1385, Vassilika Vouton, 711 10 Heraklion, Crete (Greece)


    Highlights: • Systematic research in the field of fs laser interaction with biopolymers for application in tissue engineering. • Utilizing a new biopolymer blend of collagen/elastin material for studying the interaction process in the fs domain. • Obtaining of improved, circularly shaped, interconnected nanopores, with high reproducibility from collagen/elastin layer. • Observation of randomly arranged pattern outside modification zone due to formation of an impact wave over biofilm surface. • NIH/3T3 cell-interface interaction reveal a preferable cell migration on fs laser-modified surface array. - Abstract: The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell’s responses to substrate morphology. Mice fibroblasts migration was monitored

  2. Functional roles and substrate specificities of twelve cytochromes P450 belonging to CYP52 family in n-alkane assimilating yeast Yarrowia lipolytica. (United States)

    Iwama, Ryo; Kobayashi, Satoshi; Ishimaru, Chiaki; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi


    Yarrowia lipolytica possesses twelve ALK genes, which encode cytochromes P450 in the CYP52 family. In this study, using a Y. lipolytica strain from which all twelve ALK genes had been deleted, strains individually expressing each of the ALK genes were constructed and their roles and substrate specificities were determined by observing their growth on n-alkanes and analyzing fatty acid metabolism. The results suggested that the twelve Alk proteins can be categorized into four groups based on their substrate specificity: Alk1p, Alk2p, Alk9p, and Alk10p, which have significant activities to hydroxylate n-alkanes; Alk4p, Alk5p, and Alk7p, which have significant activities to hydroxylate the ω-terminal end of dodecanoic acid; Alk3p and Alk6p, which have significant activities to hydroxylate both n-alkanes and dodecanoic acid; and Alk8p, Alk11p, and Alk12p, which showed faint or no activities to oxidize these substrates. The involvement of Alk proteins in the oxidation of fatty alcohols and fatty aldehydes was also analyzed by measuring viability of the mutant deleted for twelve ALK genes in medium containing dodecanol and by observing growth on dodecanal of a mutant strain, in which twelve ALK genes were deleted along with four fatty aldehyde dehydrogenase genes. It was suggested that ALK gene(s) is/are involved in the detoxification of dodecanol and the assimilation of dodecanal. These results imply that genes encoding CYP52-family P450s have undergone multiplication and diversification in Y. lipolytica for assimilation of various hydrophobic compounds.

  3. Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibens DSM 17395 (archetype of the marine Roseobacter clade). (United States)

    Drüppel, Katharina; Hensler, Michael; Trautwein, Kathleen; Koßmehl, Sebastian; Wöhlbrand, Lars; Schmidt-Hohagen, Kerstin; Ulbrich, Marcus; Bergen, Nils; Meier-Kolthoff, Jan P; Göker, Markus; Klenk, Hans-Peter; Schomburg, Dietmar; Rabus, Ralf


    Combining omics and enzymatic approaches, catabolic routes of nine selected amino acids (tryptophan, phenylalanine, methionine, leucine, isoleucine, valine, histidine, lysine and threonine) were elucidated in substrate-adapted cells of Phaeobacter inhibens DSM 17395 (displaying conspicuous morphotypes). The catabolic network [excluding tricarboxylic acid (TCA) cycle] was reconstructed from 71 genes (scattered across the chromosome; one-third newly assigned), with 69 encoded proteins and 20 specific metabolites identified, and activities of 10 different enzymes determined. For example, Ph. inhibens DSM 17395 does not degrade lysine via the widespread saccharopine pathway but might rather employ two parallel pathways via 5-aminopentanoate or 2-aminoadipate. Tryptophan degradation proceeds via kynurenine and 2-aminobenzoate; the latter is metabolized as known from Azoarcus evansii. Histidine degradation is analogous to the Pseudomonas-type Hut pathway via N-formyl-l-glutamate. For threonine, only one of the three genome-predicted degradation pathways (employing threonine 3-dehydrogenase) is used. Proteins of the individual peripheral degradation sequences in Ph. inhibens DSM 17395 were apparently substrate-specifically formed contrasting the non-modulated TCA cycle enzymes. Comparison of genes for the reconstructed amino acid degradation network in Ph. inhibens DSM 17395 across 27 other complete genomes of Roseobacter clade members revealed most of them to be widespread among roseobacters.

  4. Crystal Structure of the Homo sapiens Kynureninase-3-Hydroxyhippuric Acid Inhibitor Complex: Insights into the Molecular Basis Of Kynureninase Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Lima,Santiago; Kumar,Sunil; Gawandi,Vijay; Momany,Cory; Phillips,Robert S.; (Georgia)


    Homo sapiens kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic cleavage of 3-hydroxykynurenine to yield 3-hydroxyanthranilate and L-alanine as part of the tryptophan catabolic pathway leading to the de novo biosynthesis of NAD{sup +}. This pathway results in quinolinate, an excitotoxin that is an NMDA receptor agonist. High levels of quinolinate have been correlated with the etiology of neurodegenerative disorders such as AIDS-related dementia and Alzheimer's disease. We have synthesized a novel kynureninase inhibitor, 3-hydroxyhippurate, cocrystallized it with human kynureninase, and solved the atomic structure. On the basis of an analysis of the complex, we designed a series of His-102, Ser-332, and Asn-333 mutants. The H102W/N333T and H102W/S332G/N333T mutants showed complete reversal of substrate specificity between 3-hydroxykynurenine and L-kynurenine, thus defining the primary residues contributing to substrate specificity in kynureninases.

  5. A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases. (United States)

    Park, Yong Bum; Cosgrove, Daniel J


    Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this "tethered network" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.

  6. Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

    Directory of Open Access Journals (Sweden)

    Hong Wa Yung

    Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

  7. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David, E-mail: [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)


    Crystal structures of the 6s-98S FIV protease chimera with darunavir and lopinavir bound have been determined at 1.7 and 1.8 Å resolution, respectively. A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC{sub 50} values in the nanomolar range.

  8. The Broad Foundations, 2006 (United States)

    Broad Foundation, 2006


    The mission of the Broad Foundations is to transform K-12 urban public education through better governance, management, labor relations and competition; make significant contributions to advance major scientific and medical research; foster public appreciation of contemporary art by increasing access for audiences worldwide; and lead and…

  9. Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide. (United States)

    Hamm, Michelle L; McFadden, Emily J; Ghio, Michael; Lindell, Maria A M; Gerien, Kenneth S; O'Handley, Suzanne F


    The bacterial repair enzyme MutT hydrolyzes the damaged nucleotide OdGTP (the 5'-triphosphate derivative of 8-oxo-2'-deoxyguanosine; OdG), which is a known mutagen and has been linked to antibacterial action. Previous work has indicated important roles for the C8-oxygen, N7-hydrogen, and C2-exocyclic amine during OdGTP recognition by MutT. In order to gain a more nuanced understanding of the contribution of these three sites to the overall activity of MutT, we determined the reaction parameters for dGTP, OdGTP, and nine of their analogues using steady state kinetics. Our results indicate that overall high reaction efficiencies can be achieved despite altering any one of these sites. However, altering two or more sites leads to a significant decrease in efficiency. The data also suggest that, similar to another bacterial OdG repair enzyme, MutM, a specific carbonyl in the enzyme can not only promote activity by forming an active site hydrogen bond with the N7-hydrogen of OdGTP, but can also hinder activity through electrostatic repulsion with the N7-lone pair of dGTP.

  10. An in silico analysis of T-box regulated genes and T-box evolution in prokaryotes, with emphasis on prediction of substrate specificity of transporters

    Directory of Open Access Journals (Sweden)

    Kleerebezem Michiel


    Full Text Available Abstract Background T-box anti-termination is an elegant and sensitive mechanism by which many bacteria maintain constant levels of amino acid-charged tRNAs. The amino acid specificity of the regulatory element is related to a so-called specifier codon and can in principle be used to guide the functional annotation of the genes controlled via the T-box anti-termination mechanism. Results Hidden Markov Models were defined to search the T-box regulatory element and were applied to all completed prokaryotic genomes. The vast majority of the genes found downstream of the retrieved elements encoded functionalities related to transport and synthesis of amino acids and the charging of tRNA. This is completely in line with findings reported in literature and with the proposed biological role of the regulatory element. For several species, the functional annotation of a large number of genes encoding proteins involved in amino acid transport could be improved significantly on basis of the amino acid specificity of the identified T-boxes. In addition, these annotations could be extrapolated to a larger number of orthologous systems in other species. Analysis of T-box distribution confirmed that the element is restricted predominantly to species of the phylum Firmicutes. Furthermore, it appeared that the distribution was highly species specific and that in the case of amino acid transport some boxes seemed to "pop-up" only recently. Conclusion We have demonstrated that the identification of the molecular specificity of a regulatory element can be of great help in solving notoriously difficult annotation issues, e.g. by defining the substrate specificity of genes encoding amino acid transporters on basis of the amino acid specificity of the regulatory T-box. Furthermore, our analysis of the species-dependency of the occurrence of specific T-boxes indicated that these regulatory elements propagate in a semi-independent way from the genes that they control.

  11. Reactions of the cumyloxyl and benzyloxyl radicals with tertiary amides. Hydrogen abstraction selectivity and the role of specific substrate-radical hydrogen bonding. (United States)

    Salamone, Michela; Milan, Michela; DiLabio, Gino A; Bietti, Massimo


    A time-resolved kinetic study in acetonitrile and a theoretical investigation of hydrogen abstraction reactions from N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA) by the cumyloxyl (CumO(•)) and benzyloxyl (BnO(•)) radicals was carried out. CumO(•) reacts with both substrates by direct hydrogen abstraction. With DMF, abstraction occurs from the formyl and N-methyl C-H bonds, with the formyl being the preferred abstraction site, as indicated by the measured kH/kD ratios and by theory. With DMA, abstraction preferentially occurs from the N-methyl groups, whereas abstraction from the acetyl group represents a minor pathway, in line with the computed C-H BDEs and the kH/kD ratios. The reactions of BnO(•) with both substrates were best described by the rate-limiting formation of hydrogen-bonded prereaction complexes between the BnO(•) α-C-H and the amide oxygen, followed by intramolecular hydrogen abstraction. This mechanism is consistent with the very large increases in reactivity measured on going from CumO(•) to BnO(•) and with the observation of kH/kD ratios close to unity in the reactions of BnO(•). Our modeling supports the different mechanisms proposed for the reactions of CumO(•) and BnO(•) and the importance of specific substrate/radical hydrogen bond interactions, moreover providing information on the hydrogen abstraction selectivity.

  12. High Resolution Structures of Periplasmic Glucose-binding Protein of Pseudomonas putida CSV86 Reveal Structural Basis of Its Substrate Specificity. (United States)

    Pandey, Suman; Modak, Arnab; Phale, Prashant S; Bhaumik, Prasenjit


    Periplasmic substrate-binding proteins (SBPs) bind to the specific ligand with high affinity and mediate their transport into the cytoplasm via the cognate inner membrane ATP-binding cassette proteins. Because of low sequence identities, understanding the structural basis of substrate recognition by SBPs has remained very challenging. There are several structures available for the ligand-bound sugar SBPs, but very few unliganded structures are reported. No structural data are available for sugar SBPs fromPseudomonassp. to date. This study reports the first high resolution crystal structures of periplasmic glucose-binding protein fromPseudomonas putidaCSV86 (ppGBP) in unliganded form (2.5 Å) and complexed with glucose (1.25 Å) and galactose (1.8 Å). Asymmetric domain closure of ppGBP was observed upon substrate binding. The ppGBP was found to have an affinity of ∼ 0.3 μmfor glucose. The structural analysis showed that the sugars are bound to the protein mainly by hydrogen bonds, and the loss of two strong hydrogen bonds between ppGBP and galactose compared with glucose may be responsible for lowering its affinity toward galactose. The higher stability of ppGBP-glucose complex was also indicated by an 8 °C increase in the melting temperature compared with unliganded form and ppGBP-galactose complex. ppGBP binds to monosaccharide, but the structural features revealed it to have an oligosaccharide-binding protein fold, indicating that during evolution the sugar binding pocket may have undergone structural modulation to accommodate monosaccharide only.

  13. Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING. (United States)

    Khan, Irfan; Crouch, Jack D; Bharti, Sanjay Kumar; Sommers, Joshua A; Carney, Sean M; Yakubovskaya, Elena; Garcia-Diaz, Miguel; Trakselis, Michael A; Brosh, Robert M


    Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

  14. Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.

    Directory of Open Access Journals (Sweden)

    Daniel Reinecke

    Full Text Available As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP and guanosine 3',5'-cyclic monophosphate (cGMP play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP and uridine 3',5'-cyclic monophosphate (cUMP also act as second messengers. Hydrolysis by phosphodiesterases (PDEs is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.

  15. Opposing signaling of ROCK1 and ROCK2 determines the switching of substrate specificity and the mode of migration of glioblastoma cells. (United States)

    Mertsch, Sonja; Thanos, Solon


    Despite current advances in therapy, the prognosis of patients with glioblastoma has not improved sufficiently in recent decades. This is due mainly to the highly invasive capacity of glioma cells. Little is known about the mechanisms underlying this particular characteristic. While the Rho-kinase (ROCK)-dependent signaling pathways involved in glioma migration have yet to be determined, they show promise as one of the candidates in targeted glioblastoma therapy. There are two ROCK isoforms: ROCK1, which is upregulated in glioblastoma tissue compared to normal brain tissue, and ROCK2, which is also expressed in normal brain tissue. Blockage of both of these ROCK isoforms with pharmacologic inhibitors regulates the migration process. We examined the activities of ROCK1 and ROCK2 using knockdown cell lines and the newly developed stripe assay. Selective knockdown of either ROCK1 or ROCK2 exerted antidromic effects on glioma migration: while ROCK1 deletion altered the substrate-dependent migration, deletion of ROCK2 did not. Furthermore, ROCK1 knockdown reduced cell proliferation, whereas ROCK2 knockdown enhanced it. Along the signaling pathways, key regulators of the ROCK pathway are differentially affected by ROCK1 and ROCK2. These data suggest that the balanced activation of ROCKs is responsible for the substrate-specific migration and the proliferation of glioblastoma cells.

  16. [cDNA cloning, expression and determination of substrate specificity of mice selenocysteine-containing protein SelV (Selenoprotein V)]. (United States)

    Varlamova, E G; Novoselov, S V; Novoselov, V I


    To date various bioinformatics tools allowed to identify 25 selenocysteine-containing mammalian proteins. The name of these proteins assumes that they contain the amino acid selenocysteine (Sec). Functionally characterized selenocysteine-containing proteins are oxidoreductases with various functions, including glutathione peroxidases, thioredoxin reductases, deiodinases etc. However, the functions of more than half of identified proteins are still unclear, and mammalian selenoprotein SeIV is among them. We studied the selV in all stages of postnatal development with the maximum level of mRNA expression during puberty, whereas in adult mice (8-18 months) we observed a gradual decrease of expression. In order to get closer to the functional role of Selenoprotein V, we have carried out experiments on the substrate specificity and enzymatic activity measurement of this selenocysteine-containing protein. It was shown that SelV posseses glutathionperoxidase and thioredoxinreductase activities.

  17. Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Bak-Jensen, K.S.; André, G.; Gottschalk, T.E.;


    The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites - 6 and +4 ( substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved in...

  18. Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization

    Directory of Open Access Journals (Sweden)

    Klimacek Mario


    Full Text Available Abstract Background In spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from Candida tenuis in mutated (NADH-preferring form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate qxylose (g xylose/g dry cell weight/h of 0.08. The study presented herein was performed with the aim of analysing (external factors that limit qxylose of BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose. Results BP10001 and BP000, expressing C. tenuis xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L were converted sequentially, the corresponding qsubstrate values being similar for each strain (glucose: 3.0; xylose: 0.05. The distribution of fermentation products from glucose was identical for both strains whereas when using xylose, BP10001 showed enhanced ethanol yield (BP10001 0.30 g/g; BP000 0.23 g/g and decreased yields of xylitol (BP10001 0.26 g/g; BP000 0.36 g/g and glycerol (BP10001 0.023 g/g; BP000 0.072 g/g as compared

  19. Molecular cloning of a bifunctional beta-xylosidase/alpha-L-arabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme. (United States)

    Xiong, Jin-Song; Balland-Vanney, Maud; Xie, Zhi-Ping; Schultze, Michael; Kondorosi, Adam; Kondorosi, Eva; Staehelin, Christian


    Glycoside hydrolases are often members of a multigene family, suggesting individual roles for each isoenzyme. Various extracellular glycoside hydrolases have an important but poorly understood function in remodelling the cell wall during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa (Medicago sativa L.) belonging to the glycoside hydrolase family 3 (beta-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the control of the CaMV 35S promoter was expressed in the model legume Medicago truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants exhibited 5-8-fold increased activities towards three p-nitrophenyl (PNP) glycosides, namely PNP-beta-D-xyloside, PNP-alpha-L-arabinofuranoside, and PNP-alpha-L-arabinopyranoside. An antiserum raised against a synthetic peptide recognized MsXyl1, which was processed to a 65 kDa form. To characterize the substrate specificity of MsXyl1, the recombinant protein was purified from transgenic M. truncatula leaves by concanavalin A and anion chromatography. MsXyl1cleaved beta-1,4-linked D-xylo-oligosaccharides and alpha-1,5-linked L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a multifunctional beta-xylosidase/alpha-L-arabinofuranosidase/alpha-L-arabinopyranosidase implicated in cell wall turnover of arabinose and xylose, particularly in rapidly growing root tips. Moreover, the findings of this study demonstrate that stable transgenic M. truncatula plants serve as an excellent expression system for purification and characterization of proteins.

  20. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A. (Sungkyunkwan); (UTSMC)


    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  1. Influenza virus antigenicity and broadly neutralizing epitopes. (United States)

    Air, Gillian M


    A vaccine formulation that would be effective against all strains of influenza virus has long been a goal of vaccine developers, but antibodies after infection or vaccination were seen to be strain specific and there was little evidence of cross-reactive antibodies that neutralized across subtypes. Recently a number of broadly neutralizing monoclonal antibodies have been characterized. This review describes the different classes of broadly neutralizing antibodies and discusses the potential of their therapeutic use or for design of immunogens that induce a high proportion of broadly neutralizing antibodies.

  2. Insight into Substrate Preference of Two Chimeric Esterases by Combining Experiment and Molecular Simulation

    Institute of Scientific and Technical Information of China (English)

    ZHOU Xiao-li; HAN Wei-wei; ZHENG Bai-song; FENG Yan


    Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development.For this purpose,two chimeric esterases AAM7 and PAR,constructed via domain swapping between two ancient thermophilic esterases,were investigated on their molecular simulation(including homology modeling,substrates docking and substrate binding affinity validation) and enzymatic assay(specific activities and activation energies calculating).Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated,the substrate binding domains or binding pockets are important but not the only factor for substrate preference.

  3. Compound-specific 15N analysis of amino acids in 15N tracer experiments provide an estimate of newly synthesised soil protein from inorganic and organic substrates (United States)

    Charteris, Alice; Michaelides, Katerina; Evershed, Richard


    Organic N concentrations far exceed those of inorganic N in most soils and despite much investigation, the composition and cycling of this complex pool of SOM remains poorly understood. A particular problem has been separating more recalcitrant soil organic N from that actively cycling through the soil system; an important consideration in N cycling studies and for the soil's nutrient supplying capacity. The use of 15N-labelled substrates as stable isotope tracers has contributed much to our understanding of the soil system, but the complexity and heterogeneity of soil organic N prevents thorough compound-specific 15N analyses of organic N compounds and makes it difficult to examine any 15N-labelled organic products in any detail. As a result, a significant proportion of previous work has either simply assumed that since the majority of soil N is organic, all of the 15N retained in the soil is organic N (e.g. Sebilo et al., 2013) or subtracted 15N-labelled inorganic compounds from bulk values (e.g. Pilbeam et al., 1997). While the latter approach is more accurate, these methods only provide an estimate of the bulk 15N value of an extremely complex and non-uniformly labelled organic pool. A more detailed approach has been to use microbial biomass extraction (Brookes et al., 1985) and subsequent N isotopic analysis to determine the 15N value of biomass-N, representing the fraction of 15N assimilated by microbes or the 15N cycling through the 'living' or 'active' portion of soil organic N. However, this extraction method can only generate estimates and some lack of confidence in its validity and reliability remains. Here, we present an alternative technique to obtain a measure of the assimilation of an applied 15N substrate by the soil microbial biomass and an estimate of the newly synthesized soil protein, which is representative of the magnitude of the active soil microbial biomass. The technique uses a stable isotope tracer and compound-specific 15N analysis, but

  4. Broad-spectrum antiviral therapeutics.

    Directory of Open Access Journals (Sweden)

    Todd H Rider

    Full Text Available Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA Activated Caspase Oligomerizer (DRACO that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered.

  5. The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans. (United States)

    Nisha, M; Satyanarayana, T


    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.

  6. A novel sucrose hydrolase from the bombycoid silkworms Bombyx mori, Trilocha varians, and Samia cynthia ricini with a substrate specificity for sucrose. (United States)

    Wang, Huabing; Kiuchi, Takashi; Katsuma, Susumu; Shimada, Toru


    Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori, Trilocha varians, and Samia cynthia ricini and named them BmSuh, TvSuh, and ScSuh, respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori. These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry.

  7. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S. (GSK)


    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  8. Reactor for biological elimination of poorly degradable hydrocarbons by adjustment of substrate-specific retention times; Reaktor zur biologischen Eliminierung schwer abbaubarer Kohlenwasserstoffe durch Einstellung substratspezifischer Verweilzeiten

    Energy Technology Data Exchange (ETDEWEB)

    Brambach, R.


    Industrial waste water cleaning increasingly makes use of biological processes based on immobilized biomass, in order to ensure the aimed elimination of toxic substances. A novel, efficient reactor technique was developed, by which dangerous, poorly degradable dissolved hydrocarbons are selectively retained in the bioreactor thanks to an integrated membrane, while other substances are quickly discharged from the system via the effluent. With a synthetic waste water, this selective retention method achieved substrate-specific retention times which were five to fifty times higher than those of a merely hydrodynamically operated reactor system. In addition, the decoupling of high reactor and low effluent concentrations results in a higher biochemical activity in the reactor in comparison with conventional process techniques. (orig.) [Deutsch] In der industriellen Abwasserreinigung werden verstaerkt Bioverfahrenstechniken mit immobilisierter Biomasse eingesetzt, um eine gezielte Eliminierung gefaehrlicher Stoffe durchzufuehren. - Hierzu wurde eine neuartige leistungsfaehige Reaktortechnik entwickelt, die mittels einer im Bioreaktor integrierten Membran gefaehrliche, schwer abbaubare geloeste Kohlenwasserstoffe selektiv im Bioreaktor zurueckhaelt, waehrend andere Stoffe das System schnell ueber den Ablauf verlassen. Durch den stoffselektiven Rueckhalt wurden an einem synthetischen Abwasser substratspezifische Verweilzeiten erzielt, die um den Faktor 5 bis 50 ueber denen eines rein hydrodynamisch bestimmten Reaktorsystems liegen. Darueber hinaus wird aufgrund der Entkopplung von hoher Reaktor- und niedriger Ablaufkonzentration eine im Vergleich zu herkoemmlichen Verfahrenstechniken hoehere biochemische Aktivitaet im Reaktor erzielt. (orig.)

  9. Probing the Mechanism of the Mycobacterium tuberculosis [beta]-Ketoacyl-Acyl Carrier Protein Synthase III mtFabH: Factors Influencing Catalysis and Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent; Lindenberg, Sandra; Dover, Lynn G.; Sacchettini, James C.; Besra, Gurdyal S. (TAM); (Birmingham); (CNRS)


    Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.

  10. Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model (United States)

    Saleh, Mounir R.

    Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

  11. Validation, automatic generation and use of broad phonetic transcriptions

    NARCIS (Netherlands)

    Bael, Cristophe Patrick Jan Van


    Broad phonetic transcriptions represent the pronunciation of words as strings of characters from specifically designed symbol sets. In everyday life, broad phonetic transcriptions are often used as aids to pronounce (foreign) words. In addition, broad phonetic transcriptions are often used for lingu

  12. Integrated Broadband Bowtie Antenna on Transparent Silica Substrate

    CERN Document Server

    Zhang, Xingyu; Wang, Shiyi; Subbaraman, Harish; Pan, Zeyu; Zhan, Qiwen; Chen, Ray T


    The bowtie antenna is a topic of growing interest in recent years. In this paper, we design, fabricate, and characterize a modified gold bowtie antenna integrated on a transparent silica substrate. The bowtie antenna is designed with broad RF bandwidth to cover the X-band in the electromagnetic spectrum. We numerically investigate the antenna characteristics, specifically its resonant frequency and enhancement factor. Our designed bowtie antenna provides a strong broadband electric field enhancement in its feed gap. Taking advantage of the low-k silica substrate, high enhancement factor can be achieved without the unwanted reflection and scattering from the backside silicon handle which is the issue of using an SOI substrate. We simulate the dependence of resonance frequency on bowtie geometry, and verify the simulation results through experimental investigation, by fabricating different sets of bowtie antennas on silica substrates and then measuring their resonance frequencies. In addition, the far-field rad...

  13. Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis. (United States)

    Weijland, A; Parlato, G; Parmeggiani, A


    Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C). With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: possible implications for the HI-6 antidote substrate specificity. (United States)

    Artursson, Elisabet; Andersson, Per Ola; Akfur, Christine; Linusson, Anna; Börjegren, Susanne; Ekström, Fredrik


    Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

  15. Interleukin 1β Regulation of the System xc- Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR. (United States)

    Shi, Jingxue; He, Yan; Hewett, Sandra J; Hewett, James A


    System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

  16. Medically Relevant Acinetobacter Species Require a Type II Secretion System and Specific Membrane-Associated Chaperones for the Export of Multiple Substrates and Full Virulence. (United States)

    Harding, Christian M; Kinsella, Rachel L; Palmer, Lauren D; Skaar, Eric P; Feldman, Mario F


    Acinetobacter baumannii, A. nosocomialis, and A. pittii have recently emerged as opportunistic human pathogens capable of causing severe human disease; however, the molecular mechanisms employed by Acinetobacter to cause disease remain poorly understood. Many pathogenic members of the genus Acinetobacter contain genes predicted to encode proteins required for the biogenesis of a type II secretion system (T2SS), which have been shown to mediate virulence in many Gram-negative organisms. Here we demonstrate that Acinetobacter nosocomialis strain M2 produces a functional T2SS, which is required for full virulence in both the Galleria mellonella and murine pulmonary infection models. Importantly, this is the first bona fide secretion system shown to be required for virulence in Acinetobacter. Using bioinformatics, proteomics, and mutational analyses, we show that Acinetobacter employs its T2SS to export multiple substrates, including the lipases LipA and LipH as well as the protease CpaA. Furthermore, the Acinetobacter T2SS, which is found scattered amongst five distinct loci, does not contain a dedicated pseudopilin peptidase, but instead relies on the type IV prepilin peptidase, reinforcing the common ancestry of these two systems. Lastly, two of the three secreted proteins characterized in this study require specific chaperones for secretion. These chaperones contain an N-terminal transmembrane domain, are encoded adjacently to their cognate effector, and their disruption abolishes type II secretion of their cognate effector. Bioinformatic analysis identified putative chaperones located adjacent to multiple previously known type II effectors from several Gram-negative bacteria, which suggests that T2SS chaperones constitute a separate class of membrane-associated chaperones mediating type II secretion.

  17. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

    DEFF Research Database (Denmark)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig;


    to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has......-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number......Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity...

  18. Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase. Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme. (United States)

    Adolph, H W; Maurer, P; Schneider-Bernlöhr, H; Sartorius, C; Zeppezauer, M


    1. The steady-state parameters kcat and Km and the rate constants of hydride transfer for the substrates isopropanol/acetone; (S)-2-butanol, (R)-2-butanol/2-butanone; (S)-2-pentanol, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(II)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(II)LADH. 2. A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E in equilibrium E.NAD in equilibrium E.NAD.R1R2 CHOH in equilibrium E.NADH.R1R2CO in equilibrium E.NADH in equilibrium E. 3. On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions. 2-Butanone and 2-pentanone reductions revealed (S)-2-butanol (86%) and (S)-2-pentanol (95%) as the major products. 4. The observed enantioselectivity implies the existence of two productive ternary complexes; E.NADH.(pro-S) 2-butanone and E.NADH.(pro-R) 2-butanone. All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined. These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent kcat/Km values for the two different ternary-complex configurations of 2-butanone. Additionally, these data have been used for computer simulations of the corresponding reaction cycles. Calculated, simulated and experimental data were found to be in good agreement. Thus, the system (S)-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADH-catalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism. 5. The effects of Co(II) substitution on the

  19. The 1.6 A crystal structure of pyranose dehydrogenase from Agaricus meleagris rationalizes substrate specificity and reveals a flavin intermediate.

    Directory of Open Access Journals (Sweden)

    Tien Chye Tan

    Full Text Available Pyranose dehydrogenases (PDHs are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O activates oxygen by a mechanism that proceeds via a covalent flavin C(4a-hydroperoxide intermediate. Although the flavin C(4a adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2

  20. Specific features of sputtered atoms transport during Ta sub 2 O sub 5 film deposition on substrates of complicated spatial configuration

    CERN Document Server

    Bystrov, Y A; Volpyas, V A; Govako, E A; Timofeev, D E; Troshkov, V V


    The possibility of providing for uniform properties of the Ta sub 2 O sub 5 films on the surface of substrates, having complex form, is considered. The attempt is made to consider the sputtered atoms transport in the space of the target-substrate drift through the determination of the thermalization zone geometrical sizes and the subsequent transition of the sputtered particles into the diffusion mode. Various calculational models and static modeling methods are used for describing the process of the sputtered particles transport

  1. Broad Frequency LTCC Vertical Interconnect Transition for Multichip Modules and System on Package Applications (United States)

    Decrossas, Emmanuel; Glover, Michael D.; Porter, Kaoru; Cannon, Tom; Mantooth, H. Alan; Hamilton, M. C.


    Various stripline structures and flip chip interconnect designs for high-speed digital communication systems implemented in low temperature co-fired ceramic (LTCC) substrates are studied in this paper. Specifically, two different transition designs from edge launch 2.4 millimeter connectors to stripline transmission lines embedded in LTCC are discussed. After characterizing the DuPont (sup trademark) 9K7 green tape, different designs are proposed to improve signal integrity for high-speed digital data. The full-wave simulations and experimental data validate the presented designs over a broad frequency band from Direct Current to 50 gigahertz and beyond.

  2. Substrate Specificity of the Bacillus subtilis BY-Kinase PtkA Is Controlled by Alternative Activators: TkmA and SalA

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Shi, Lei; Kalantari, Aida;


    Bacterial protein-tyrosine kinases (BY-kinases) are known to regulate different aspects of bacterial physiology, by phosphorylating cellular protein substrates. Physiological cues that trigger BY-kinases activity are largely unexplored. In Proteobacteria, BY-kinases contain a cytosol-exposed cata...

  3. Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances. (United States)

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi


    Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality.

  4. Advance in identification and analysis of epitope specificities of broadly neutralizing anti-HIV-1 sera%抗HIV广谱中和血清表位特异性研究进展

    Institute of Scientific and Technical Information of China (English)

    胡新韬; 洪坤学; 邵一鸣


    HIT疫苗研究的巨大障碍在于目前设计的包含Env蛋白或表住的疲苗在临床前及临床试验中难于诱导产生广谱中和抗体.近来的研究表明一些HIV-1感染者血清中存在的抗体能够中和多种病毒株,而新的表位作图技术对这些广谱中和血清表位特异性的深入分析将对广谱中和抗体靶向的病毒表位提供新的线索.通过广谱中和血清表位特异性研究获得的信息将促进新的广谱中和单克隆抗体的分离和潜在新表住的鉴定,为合理设计新的疫苗免疫原提供关键信息.%One obstacle to the development of an effective HIV-1 vaccine has been the difficulty in inducing broadly neutralizing antibodies with protective functions. However, recent studies show that antibodies in the sera of some HIV-1 infected individuals can neutralize diverse HIV-1 isolates. Detailed analyses of these sera with recently developed sophistical mapping methods can provide new insights into the viral epitopes targeted by broadly reactive NAbs. The new information emerging from these mapping efforts may be conducive to sharpening efforts to isolate new bnmAbs and moreover, may provide crucial information for the rational design of novel vaccine candidates.

  5. Offshore Substrate (United States)

    California Department of Resources — This shapefile displays the distribution of substrate types from Pt. Arena to Pt. Sal in central/northern California. Originally this data consisted of seven paper...

  6. Purification and substrate specificity of beta-xylosidase from sycamore cell (Acer pseudoplatanus L.): application for structural analysis of xylose-containing N-linked oligosaccharides. (United States)

    Tezuka, K; Hayashi, M; Ishihara, H; Nishimura, M; Onozaki, K; Takahashi, N


    A beta-xylosidase was purified 51-fold from culture medium of sycamore (Acer pseudoplatanus L.) cells using p-nitrophenyl beta-D-xylopyranoside as a substrate. This enzyme can remove a xylose residue from asparagine-linked oligosaccharides, derivatized with 2-aminopyridine. A pentasaccharide, Xy1 beta 2Man beta 4GlcNAc beta 4(Fuc-alpha 3)GlcNAc was the favorite substrate in N-linked oligosaccharides, but a xylose residue in Xy1 beta 2(Man-alpha 3)Man beta sequence could not be removed by the enzyme. We also propose an efficient method for detection of xylose residue in N-linked oligosaccharides by a combination of the two-dimensional sugar mapping technique and the xylosidase digestion.

  7. Broad-spectrum antiviral agents

    Directory of Open Access Journals (Sweden)

    Jun-Da eZhu


    Full Text Available Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents.

  8. Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution. (United States)

    Desai, Bijoy J; Goto, Yuki; Cembran, Alessandro; Fedorov, Alexander A; Almo, Steven C; Gao, Jiali; Suga, Hiroaki; Gerlt, John A


    Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼10(17)) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼10(2)) to the 10(17) rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

  9. Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

    KAUST Repository

    Witz, Sandra


    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.

  10. Broad resonances in transport theory

    CERN Document Server

    Leupold, S


    The extension of the transport theoretical framework to include states with a broad mass distribution is discussed. The proper life-time and cross sections for a state with an arbitrarily given invariant mass is discussed in detail. (author)

  11. Multiple alternative substrate kinetics. (United States)

    Anderson, Vernon E


    The specificity of enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme's specificity using kinetics have been utilized over the decades. Fersht's definition of the ratio kcat/Km for two different substrates as the "specificity constant" (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for enzymes obeying Michaelis-Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

  12. Substrate tolerant direct block copolymer nanolithography

    DEFF Research Database (Denmark)

    Li, Tao; Wang, Zhongli; Schulte, Lars


    simplifies the main stream BC lithography process, showing a broad substrate tolerance and allowing for efficient pattern transfer over wafer scale. PDMS-rich poly(styrene-b-dimethylsiloxane) (PS-b-PDMS) copolymers are directly applied on substrates including polymers, silicon and graphene. A single oxygen...

  13. Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.). (United States)

    Tezuka, K; Hayashi, M; Ishihara, H; Akazawa, T; Takahashi, N


    We measured the activities of alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, beta-1,4-mannosyl-glycoprotein beta-1,2-xylosyltransferase and glycoprotein 3-alpha-L-fucosyltransferase in the Golgi fraction of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using fluorescence-labelled oligosaccharides as acceptor substrates for these transferase reactions. The structures of the pyridylaminated oligosaccharides produced by these reactions were analyzed by two-dimensional sugar mapping using high-performance liquid chromatography. We demonstrated that (formula; see text) was processed to produce by these in vitro reactions. On the basis of these results, we discuss a biosynthetic pathway for xylose containing N-linked oligosaccharides in plant glycoproteins.

  14. The Broad Autism Phenotype Questionnaire (United States)

    Hurley, Robert S. E.; Losh, Molly; Parlier, Morgan; Reznick, J. Steven; Piven, Joseph


    The broad autism phenotype (BAP) is a set of personality and language characteristics that reflect the phenotypic expression of the genetic liability to autism, in non-autistic relatives of autistic individuals. These characteristics are milder but qualitatively similar to the defining features of autism. A new instrument designed to measure the…

  15. Site-specific forest-assembly of single-wall carbon nanotubes on electron-beam patterned SiO{sub x}/Si substrates

    Energy Technology Data Exchange (ETDEWEB)

    Wei Haoyan [Materials Science and Engineering Program, Department of Chemical, Materials and Biomolecular Engineering, Institute of Materials Science, University of Connecticut, Storrs, CT 06269 (United States); Kim, Sang Nyon; Kim, Sejong [Nanomaterials Optoelectronics Laboratory, Polymer Program, Institute of Materials Science, Department of Chemistry, University of Connecticut, Storrs, CT 06269 (United States); Huey, Bryan D. [Materials Science and Engineering Program, Department of Chemical, Materials and Biomolecular Engineering, Institute of Materials Science, University of Connecticut, Storrs, CT 06269 (United States); Papadimitrakopoulos, Fotios [Nanomaterials Optoelectronics Laboratory, Polymer Program, Institute of Materials Science, Department of Chemistry, University of Connecticut, Storrs, CT 06269 (United States); Marcus, Harris L. [Materials Science and Engineering Program, Department of Chemical, Materials and Biomolecular Engineering, Institute of Materials Science, University of Connecticut, Storrs, CT 06269 (United States)], E-mail:


    Based on electron-beam direct writing on the SiO{sub x}/Si substrates, favorable absorption sites for ferric cations (Fe{sup 3+} ions) were created on the surface oxide layer. This allowed Fe{sup 3+}-assisted self-assembled arrays of single-wall carbon nanotube (SWNT) probes to be produced. Auger investigation indicated that the incident energetic electrons depleted oxygen, creating more dangling bonds around Si atoms at the surface of the SiO{sub x} layer. This resulted in a distinct difference in the friction forces from unexposed regions as measured by lateral force microscopy (LFM). Atomic force microscopy (AFM) affirmed that the irradiated domains absorbed considerably more Fe{sup 3+} ions upon immersion into pH 2.2 aqueous FeCl{sub 3} solution. This rendered a greater yield of FeO(OH)/FeOCl precipitates, primarily FeO(OH), upon subsequent washing with lightly basic dimethylformamide (DMF) solution. Such selective metal-functionalization established the basis for the subsequent patterned forest-assembly of SWNTs as demonstrated by resonance Raman spectroscopy.

  16. Novel intracellular GH10 xylanase from Cohnella laeviribosi HY-21: biocatalytic properties and alterations of substrate specificities by site-directed mutagenesis of Trp residues. (United States)

    Kim, Do Young; Han, Mi Kyoung; Oh, Hyun-Woo; Bae, Kyung Sook; Jeong, Tae-Sook; Kim, Sung Uk; Shin, Dong-Ha; Kim, In-Ho; Rhee, Young Ha; Son, Kwang-Hee; Park, Ho-Yong


    The novel intracellular GH10 xylanase (iXylC) gene (1023-bp) of Cohnella laeviribosi HY-21 encoded a protein consisting of 340 amino acids with a deduced molecular mass of 39,330Da and a calculated pI of 5.81. The primary structure of iXylC was 70% identical to that of Geobacillus sp. GH10 enzyme (GenBank accession number: EDV78425). Xylanolytic activity of the His-tagged iXylC overproduced in Escherichiacoli BL21 was stimulated by 2.2-fold in the presence of 0.5% non-ionic detergents. iXylC produced a mixture of xylooligosaccharides (xylobiose to xylooctaose) from xylotriose and xylotetraose used as the hydrolytic substrate. In addition, it exhibited considerable cleavage activities for p-nitrophenylxylopyranoside (PNP-xylopyranoside) and PNP-cellobioside, indicating that iXylC is a unique GH10 enzyme. The hydrolytic activity (57.8IUmL(-1)) of iXylC toward PNP-xylopyranoside increased to 8.3-fold by W217A and W315A mutations, while mutations of W133A, W295A, and W303A abolished the hydrolytic activity of the enzyme.

  17. Substrate Handbook for Biogas Production; Substrathandbok foer biogasproduktion

    Energy Technology Data Exchange (ETDEWEB)

    Carlsson, My; Uldal, Martina (AnoxKaldnes AB, Lund (Sweden))


    Today, co-digestion plants in Sweden treat a broad range of different substrates, of which some have not previously been used for anaerobic digestion. The major part of this organic waste derives from households, restaurants, food industries and farms. When evaluating a new substrate as feed for anaerobic digestion, several different aspects need to be taken into consideration, such as anaerobic degradability, TS/VS content, nutrient composition and risk for mechanical problems. Consequently, there is a need for practical guidelines on how to evaluate new substrates as raw materials for biogas production, including not only gas yield but also what practical and microbiological problems that may arise when the specific substrate is treated together with other substrates in the plant. The aim with this handbook is to provide a basis on how to evaluate new substrates as feed for anaerobic digestion. The intention is that this material will save time and effort for the personnel at the plant when they come in contact with new types of waste. Also, the aim is to facilitate the process of identifying new substrates within the ABP-regulation (1774/2002) and what requirements are then demanded on handling. The work with the handbook has been divided in three different parts; (1) an extensive literature study and a compilation of the achieved results, (2) interviews with personnel at most of the Swedish co-digestion plants to identify substrates and problems of interest, and (3) lab tests of selected substrates. The lab tests included Bio Methane Potential (BMP) tests as well as a simple characterization of each substrate based on fat/protein/carbohydrate content. All data origins from anaerobic digestion within the mesophilic temperature range, but the results and discussion are applicable also for thermophilic anaerobic digestion. The result of this work is a written report together with an Excel file which are to be directly used by the biogas plants as a basis in the

  18. Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences. (United States)

    Hebert, Daniel N; Clerico, Eugenia M; Gierasch, Lila M


    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.

  19. Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases. (United States)

    Chahinian, Henri; Ali, Yassine Ben; Abousalham, Abdelkarim; Petry, Stefan; Mandrich, Luigi; Manco, Guiseppe; Canaan, Stephane; Sarda, Louis


    We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.

  20. Structural evidence of substrate specificity in mammalian peroxidases: structure of the thiocyanate complex with lactoperoxidase and its interactions at 2.4 A resolution. (United States)

    Sheikh, Ishfaq Ahmed; Singh, Amit Kumar; Singh, Nagendra; Sinha, Mau; Singh, S Baskar; Bhushan, Asha; Kaur, Punit; Srinivasan, Alagiri; Sharma, Sujata; Singh, Tej P


    The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN(-)) has been determined at 2.4A resolution. It revealed that the SCN(-) ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN(-) ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN(-) forms a hydrogen bond with a water (Wat) molecule at position 6'. This water molecule is stabilized by two hydrogen bonds with Gln(423) N(epsilon2) and Phe(422) oxygen. In contrast, the placement of the SCN(-) ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln(423), Phe(422) oxygen, and Wat(6)' in LPO is occupied primarily by the side chain of Phe(407) in MPO due to an entirely different conformation of the loop corresponding to the segment Arg(418)-Phe(431) of LPO. This arrangement in MPO does not favor a similar orientation of the SCN(-) ion. The orientation of the catalytic product OSCN(-) as reported in the structure of LPO.OSCN(-) is similar to the orientation of SCN(-) in the structure of LPO.SCN(-). Similarly, in the structure of LPO.SCN(-).CN(-), in which CN(-) binds at Wat(1), the position and orientation of the SCN(-) ion are also identical to that observed in the structure of LPO.SCN.

  1. Broad Diphotons from Narrow States

    CERN Document Server

    An, Haipeng; Zhang, Yue


    ATLAS and CMS have each reported a modest diphoton excess consistent with the decay of a broad resonance at ~ 750 GeV. We show how this signal can arise in a weakly coupled theory comprised solely of narrow width particles. In particular, if the decaying particle is produced off-shell, then the associated diphoton resonance will have a broad, adjustable width. We present simplified models which explain the diphoton excess through the three-body decay of a scalar or fermion. Our minimal ultraviolet completion is a weakly coupled and renormalizable theory of a singlet scalar plus a heavy vector-like quark and lepton. The smoking gun of this mechanism is an asymmetric diphoton peak recoiling against missing transverse energy, jets, or leptons.

  2. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. (United States)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig; Bennett, Eric P; Mandel, Ulla; Takeuchi, Hideyuki; Kato, Kentaro; Irimura, Tatsuro; Suryanarayanan, Ganesh; Hollingsworth, Michael A; Clausen, Henrik


    Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

  3. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao


    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC and endo-β-1,4-xylanase (EC The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

  4. Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G). (United States)

    Han, Ruizhi; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian


    In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of L-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2% higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.

  5. Molecular cloning and biochemical characterization of VIM-12, a novel hybrid VIM-1/VIM-2 metallo-beta-lactamase from a Klebsiella pneumoniae clinical isolate, reveal atypical substrate specificity. (United States)

    Kontou, Maria; Pournaras, Spyros; Kristo, Ioulia; Ikonomidis, Alexandros; Maniatis, Antonios N; Stathopoulos, Constantinos


    Metallo-beta-lactamases (MBLs) are considered an emerging family of Zn2+-dependent enzymes that significantly contribute to the resistance of many nosocomial pathogens against beta-lactam antimicrobials. Since these plasmid-encoded enzymes constitute specific molecular targets for beta-lactams, their exact mode of action is greatly important in deploying efficient anti-infective treatments and for the control of severe multi-resistant nosocomial infections, which becomes a global problem. A novel hybrid VIM-1/VIM-2-type beta-lactamase (named VIM-12) has recently been identified in a clinical isolate of Klebsiella pneumoniae in Greece. The sequence of this enzyme is highly similar with that of VIM-1 at its N-terminal region and with that of VIM-2 at its C-terminal region, raising the question of whether this sequence similarity reflects also a similar functional role. Moreover, the possible contribution of this novel beta-lactamase to the overall antibiotic resistance of this specific clinical isolate was investigated. The gene encoding VIM-12 was cloned and expressed, and the recombinant enzyme was used for detailed kinetic analysis, using a variety of beta-lactam antibiotics. VIM-12 was found to exhibit narrow substrate specificity, compared to other known beta-lactamases, limited mainly to penicillin and to a much lesser extent to imipenen. Interestingly, meropenem was found to act as a noncompetitive inhibitor of the enzyme, although the active site of VIM-12 exhibited complete conservation of residues among VIM enzymes. We conclude that VIM-12 represents a novel and unique member of the family of known metallo-beta-lactamases, exhibiting atypical substrate specificity.

  6. Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor. (United States)

    Fonseca-Madrigal, Jorge; Navarro, Juan C; Hontoria, Francisco; Tocher, Douglas R; Martínez-Palacios, Carlos A; Monroig, Óscar


    Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C18 PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C18 PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages.

  7. Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor[S (United States)

    Fonseca-Madrigal, Jorge; Navarro, Juan C.; Hontoria, Francisco; Tocher, Douglas R.; Martínez-Palacios, Carlos A.; Monroig, Óscar


    Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C18 PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C18 PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages. PMID:24792929

  8. Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV). (United States)

    Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall


    Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use.

  9. A Broad Specificity Nucleoside Kinase from Thermoplasma acidophilum


    Elkin, Sarah R.; Kumar, Abhinav; Price, Carol W.; Columbus, Linda


    The crystal structure of Ta0880, determined at 1.91 A resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β /α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, frutokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had bee...

  10. Conditioning-specific reflex modification of the rabbit's nictitating membrane response and heart rate: behavioral rules, neural substrates, and potential applications to posttraumatic stress disorder. (United States)

    Burhans, Lauren B; Smith-Bell, Carrie; Schreurs, Bernard G


    Interest in classical conditioning is usually focused on anticipatory responses to a stimulus associated with a significant event, and it is assumed that responses to the event itself are reflexive, involuntary, and relatively invariant. However, there is compelling evidence that both the rabbit nictitating membrane response (NMR) and heart rate response (HR), well-known reflexive reactions to aversive events, can change quite dramatically as a function of learning when measured in the absence of the conditioned stimulus. In the case of NMR conditioning, a simple blink is transformed into a larger and more complex response. For HR conditioning, reflexive heart rate acceleration can actually change to heart rate deceleration. In both cases, the reflex comes to resemble the conditioned response and follows some of the same behavioral laws. This change in response to the aversive event itself or weaker forms of that event is called conditioning-specific reflex modification (CRM). CRM may force us to reevaluate the behavioral and neural consequences of classical conditioning and may have important consequences for the treatment of conditions such as posttraumatic stress disorder.

  11. Protease activity of PprI facilitates DNA damage response: Mn2+-dependence and substrate sequence-specificity of the proteolytic reaction.

    Directory of Open Access Journals (Sweden)

    Yunguang Wang

    Full Text Available The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes' expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism.

  12. Substrate specificity and gene expression of two Penicillium chrysogenum α-L-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54. (United States)

    Sakamoto, Tatsuji; Inui, Misako; Yasui, Kana; Hosokawa, Sachiko; Ihara, Hideshi


    We previously isolated two α-L-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L-arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.

  13. Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IX. Host plant and population specific epicuticular hydrocarbon expression influences mate choice and sexual selection. (United States)

    Havens, J A; Etges, W J


    Sexual signals in cactophilic Drosophila mojavensis include cuticular hydrocarbons (CHCs), contact pheromones that mediate female discrimination of males during courtship. CHCs, along with male courtship songs, cause premating isolation between diverged populations, and are influenced by genotype × environment interactions caused by different host cacti. CHC profiles of mated and unmated adult flies from a Baja California and a mainland Mexico population of D. mojavensis reared on two host cacti were assayed to test the hypothesis that male CHCs mediate within-population female discrimination of males. In multiple choice courtship trials, mated and unmated males differed in CHC profiles, indicating that females prefer males with particular blends of CHCs. Mated and unmated females significantly differed in CHC profiles as well. Adults in the choice trials had CHC profiles that were significantly different from those in pair-mated adults from no-choice trials revealing an influence of sexual selection. Females preferred different male CHC blends in each population, but the influence of host cactus on CHC variation was significant only in the mainland population indicating population-specific plasticity in CHCs. Different groups of CHCs mediated female choice-based sexual selection in each population suggesting that geographical and ecological divergence has the potential to promote divergence in mate communication systems.

  14. S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids. (United States)

    Alves, L C; Melo, R L; Sanderson, S J; Mottram, J C; Coombs, G H; Caliendo, G; Santagada, V; Juliano, L; Juliano, M A


    We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and

  15. Ghost imaging with broad distance

    Institute of Scientific and Technical Information of China (English)

    段德洋; 张路; 杜少将; 夏云杰


    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is.

  16. A split-ubiquitin yeast two-hybrid screen to examine the substrate specificity of atToc159 and atToc132, two Arabidopsis chloroplast preprotein import receptors.

    Directory of Open Access Journals (Sweden)

    Siddhartha Dutta

    Full Text Available Post-translational import of nucleus-encoded chloroplast pre-proteins is critical for chloroplast biogenesis, and the Toc159 family of proteins serve as receptors for the process. Toc159 shares with other members of the family (e.g. Toc132, homologous GTPase (G- and Membrane (M- domains, but a highly dissimilar N-terminal acidic (A- domain. Although there is good evidence that atToc159 and atToc132 from Arabidopsis mediate the initial sorting step, preferentially recognizing photosynthetic and non-photosynthetic preproteins, respectively, relatively few chloroplast preproteins have been assigned as substrates for particular members of the Toc159 family, which has limited the proof for the hypothesis. The current study expands the number of known preprotein substrates for members of the Arabidopsis Toc159 receptor family using a split-ubiquitin membrane-based yeast two-hybrid system using the atToc159 G-domain (Toc159G, atToc132 G-domain (Toc132G and atToc132 A- plus G-domains (Toc132AG as baits. cDNA library screening with all three baits followed by pairwise interaction assays involving the 81 chloroplast preproteins identified show that although G-domains of the Toc159 family are sufficient for preprotein recognition, they alone do not confer specificity for preprotein subclasses. The presence of the A-domain fused to atToc132G (Toc132AG not only positively influences its specificity for non-photosynthetic preproteins, but also negatively regulates the ability of this receptor to interact with a subset of photosynthetic preproteins. Our study not only substantiates the fact that atToc132 can serve as a receptor by directly binding to chloroplast preproteins but also proposes the existence of subsets of preproteins with different but overlapping affinities for more than one member of the Toc159 receptor family.

  17. Broad Leaves in Strong Flow

    CERN Document Server

    Miller, Laura


    Flexible broad leaves are thought to reconfigure in the wind and water to reduce the drag forces that act upon them. Simple mathematical models of a flexible beam immersed in a two-dimensional flow will also exhibit this behavior. What is less understood is how the mechanical properties of a leaf in a three-dimensional flow will passively allow roll up into a cone shape and reduce both drag and vortex induced oscillations. In this fluid dynamics video, the flows around the leaves are compared with those of simplified sheets using 3D numerical simulations and physical models. For some reconfiguration shapes, large forces and oscillations due to strong vortex shedding are produced. In the actual leaf, a stable recirculation zone is formed within the wake of the reconfigured cone. In physical and numerical models that reconfigure into cones, a similar recirculation zone is observed with both rigid and flexible tethers. These results suggest that the three-dimensional cone structure in addition to flexibility is ...

  18. Prebiotics: preferential substrates for specific germs? (United States)

    Roberfroid, M B


    A prebiotic is "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon." The premise is based on the hypothesis that the large gut in humans contains bacteria that are beneficial or detrimental to health. Although this generalization probably gives too simplistic a view of gut microbiology, it is a feasible working concept. Currently, food components that seem to exert the best prebiotic effects are inulin-type fructans. In pure culture, most species of bifidobacteria are adapted to the utilization of these nondigestible oligosaccharides but many other bacteria are also capable of metabolizing them. Clearly, these studies of pure bacteria are of limited use unless their results are supported by the results of studies using mixed cultures. Indeed, as many components of the gut microbiota as possible should be measured to indicate a true prebiotic effect. Simple stimulation of bifidobacteria is insufficient to demonstrate an effect; the effects on other gut microorganisms in vivo with human volunteers is necessary. Adjustment of the composition and activities of the colonic microflora so that health-promoting activities are optimized remains key in functional food development. New methods are being applied extensively to human gut microbiology and promise the degree of reliability required to detect subtle changes in colonic microflora composition and to correlate such changes with health benefits. This is a review of the present state of knowledge concerning prebiotics, with emphasis on the criteria used for classification, mechanisms of selective growth stimulation, and physiologic effects.

  19. 76 FR 34087 - Broad Stakeholder Survey (United States)


    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until.... The Broad Stakeholder Survey is designed to gather stakeholder feedback on the effectiveness of...

  20. 78 FR 20119 - Broad Stakeholder Survey (United States)


    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 30-day... soliciting comments concerning the Broad Stakeholder Survey. DHS previously published this ICR in the Federal... responders across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  1. 77 FR 50144 - Broad Stakeholder Survey (United States)


    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until... across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  2. Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity.

    Directory of Open Access Journals (Sweden)

    Nathan E Kreel

    Full Text Available Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363 found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.

  3. Study of Carbon Nanotube-Substrate Interaction

    Directory of Open Access Journals (Sweden)

    Jaqueline S. Soares


    Full Text Available Environmental effects are very important in nanoscience and nanotechnology. This work reviews the importance of the substrate in single-wall carbon nanotube properties. Contact with a substrate can modify the nanotube properties, and such interactions have been broadly studied as either a negative aspect or a solution for developing carbon nanotube-based nanotechnologies. This paper discusses both theoretical and experimental studies where the interaction between the carbon nanotubes and the substrate affects the structural, electronic, and vibrational properties of the tubes.

  4. Power electronics substrate for direct substrate cooling (United States)

    Le, Khiet [Mission Viejo, CA; Ward, Terence G [Redondo Beach, CA; Mann, Brooks S [Redondo Beach, CA; Yankoski, Edward P [Corona, CA; Smith, Gregory S [Woodland Hills, CA


    Systems and apparatus are provided for power electronics substrates adapted for direct substrate cooling. A power electronics substrate comprises a first surface configured to have electrical circuitry disposed thereon, a second surface, and a plurality of physical features on the second surface. The physical features are configured to promote a turbulent boundary layer in a coolant impinged upon the second surface.

  5. It takes two to tango: two TatA paralogues and two redox enzyme-specific chaperones are involved in the localization of twin-arginine translocase substrates in Campylobacter jejuni. (United States)

    Liu, Yang-Wei; Hitchcock, Andrew; Salmon, Robert C; Kelly, David J


    The food-borne zoonotic pathogen Campylobacter jejuni has complex electron transport chains required for growth in the host, many of which contain cofactored periplasmic enzymes localized by the twin-arginine translocase (TAT). We report here the identification of two paralogues of the TatA translocase component in C. jejuni strain NCTC 11168, encoded by cj1176c (tatA1) and cj0786 (tatA2). Deletion mutants constructed in either or both of the tatA1 and tatA2 genes displayed distinct growth and enzyme activity phenotypes. For sulphite oxidase (SorAB), the multi-copper oxidase (CueO) and alkaline phosphatase (PhoX), complete dependency on TatA1 for correct periplasmic activity was observed. However, the activities of nitrate reductase (NapA), formate dehydrogenase (FdhA) and trimethylamine N-oxide reductase (TorA) were significantly reduced in the tatA2 mutant. In contrast, the specific rate of fumarate reduction catalysed by the flavoprotein subunit of the methyl menaquinone fumarate reductase (MfrA) was similar in periplasmic fractions of both the tatA1 and the tatA2 mutants and only the deletion of both genes abolished activity. Nevertheless, unprocessed MfrA accumulated in the periplasm of the tatA1 (but not tatA2) mutant, indicating aberrant signal peptide cleavage. Surprisingly, TatA2 lacks two conserved residues (Gln8 and Phe39) known to be essential in Escherichia coli TatA and we suggest it is unable to function correctly in the absence of TatA1. Finally, only two TAT chaperones (FdhM and NapD) are encoded in strain NCTC 11168, which mutant studies confirmed are highly specific for formate dehydrogenase and nitrate reductase assembly, respectively. Thus, other TAT substrates must use general chaperones in their biogenesis.

  6. Novel bottom-up SERS substrates for quantitative and parallelized analytics. (United States)

    Strelau, Katharina K; Schüler, Thomas; Möller, Robert; Fritzsche, Wolfgang; Popp, Jürgen


    Surface-enhanced Raman spectroscopy (SERS) is an emerging technology in the field of analytics. Due to the high sensitivity in connection with specific Raman molecular fingerprint information SERS can be used in a variety of analytical, bioanalytical, and biosensing applications. However, for the SERS effect substrates with metal nanostructures are needed. The broad application of this technology is greatly hampered by the lack of reliable and reproducible substrates. Usually the activity of a given substrate has to be determined by time-consuming experiments such as calibration or ultramicroscopic studies. To use SERS as a standard analytical tool, cheap and reproducible substrates are required, preferably with a characterization technique that does not interfere with the subsequent measurements. Herein we introduce an innovative approach to produce low-cost and large-scale reproducible substrates for SERS applications, which allows easy and economical production of micropatterned SERS active surfaces on a large scale. This approach is based on an enzyme-induced growth of silver nanostructures. The special structural feature of the enzymatically deposited silver nanoparticles prevents the breakdown of SERS activity even at high particle densities (particle density >60%) that lead to a conductive layer. In contrast to other approaches, this substrate exhibits a relationship between electrical conductivity and the resulting SERS activity of a given spot. This enables the prediction of the SERS activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of enzymatic silver nanoparticles on a large scale, utilizing a simple measurement of the electrical conductivity. Furthermore, through a correlation between the conductivity and the SERS activity of the substrates it is possible to quantify SERS measurements with these substrates.

  7. Broad Prize: Do the Successes Spread? (United States)

    Samuels, Christina A.


    When the Broad Prize for Urban Education was created in 2002, billionaire philanthropist Eli Broad said he hoped the awards, in addition to rewarding high-performing school districts, would foster healthy competition; boost the prestige of urban education, long viewed as dysfunctional; and showcase best practices. Over the 10 years the prize has…

  8. Direct transfer of graphene onto flexible substrates (United States)

    Pimenta, Luiz Gustavo; Song, Yi; Zeng, Tingying; Dresselhaus, Mildred; Kong, Jing; Araujo, Paulo


    We explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate. Various substrates of general interest in research and industry were studied including polytetrafluoroethylene filter membranes, PVC, cellulose nitrate/cellulose acetate filter membranes, polycarbonate, paraffin, polyethylene terephthalate, paper, and cloth. By comparing the properties of these substrates, two critical factors to ensure a successful transfer on bare substrates were identified: the substrate's hydrophobicity and good contact between the substrate and graphene. For substrates that do not satisfy those requirements, polymethylmethacrylate can be used as a surface modifier or glue to ensure successful transfer. Our results can be applied to facilitate present processes and open up directions for applications of chemical vapor deposition (CVD) graphene on flexible substrates. A broad range of applications of CVD graphene can be envisioned, including fabrication of graphene devices for opto/organic electronics, graphene membranes for gas/liquid separation, and ubiquitous electronics with graphene.

  9. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling. (United States)

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A


    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models.

  10. Measuring Prevention More Broadly, An Empirical... (United States)

    U.S. Department of Health & Human Services — Measuring Prevention More Broadly, An Empirical Assessment of CHIPRA Core Measures Differences in CHIP design and structure, across states and over time, may limit...

  11. Personal glucose meters for detection and quantification of a broad range of analytes (United States)

    Lu, Yi; Xiang, Yu


    A general methodology for the development of highly sensitive and selective sensors that can achieve portable, low-cost and quantitative detection of a broad range of targets using only a personal glucose meter (PGM) is disclosed. The method uses recognition molecules that are specific for a target agent, enzymes that can convert an enzyme substrate into glucose, and PGM. Also provided are sensors, which can include a solid support to which is attached a recognition molecule that permits detection of a target agent, wherein the recognition molecule specifically binds to the target agent in the presence of the target agent but not significantly to other agents as well as an enzyme that can catalyze the conversion of a substance into glucose, wherein the enzyme is attached directly or indirectly to the recognition molecule, and wherein in the presence of the target agent the enzyme can convert the substance into glucose. The disclosed sensors can be part of a lateral flow device. Methods of using such sensors for detecting target agents are also provided.

  12. Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways. (United States)

    Dang, Michelle; Armbruster, Nicole; Miller, Miles A; Cermeno, Efrain; Hartmann, Monika; Bell, George W; Root, David E; Lauffenburger, Douglas A; Lodish, Harvey F; Herrlich, Andreas


    Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.

  13. Giant Broad Line Regions in Dwarf Seyferts

    CERN Document Server

    Devereux, Nick


    High angular resolution spectroscopy obtained with the Hubble Space Telescope (HST) has revealed a remarkable population of galaxies hosting dwarf Seyfert nuclei with an unusually large broad-line region (BLR). These objects are remarkable for two reasons. Firstly, the size of the BLR can, in some cases, rival those seen in the most luminous quasars. Secondly, the size of the BLR is not correlated with the central continuum luminosity, an observation that distinguishes them from their reverberating counterparts. Collectively, these early results suggest that non-reverberating dwarf Seyferts are a heterogeneous group and not simply scaled versions of each other. Careful inspection reveals broad H Balmer emission lines with single peaks, double peaks, and a combination of the two, suggesting that the broad emission lines are produced in kinematically distinct regions centered on the black hole (BH). Because the gravitational field strength is already known for these objects, by virtue of knowing their BH mass, ...

  14. Modelling Demand for Broad Money in Australia


    Abbas Valadkhani


    The existence of a stable demand for money is very important for the conduct of monetary policy. It is argued that previous work on the demand for money in Australia has not been very satisfactory in a number of ways. This paper examines the long- and short-run determinants of the demand for broad money employing the Johansen cointegration technique and a short-run dynamic model. Using quarterly data for the period 1976:3-2002:2, this paper finds, inter alia, that the demand for broad money i...

  15. Ketone Bodies as Brain Substrates


    Silva, Paula Sofia Valente da


    SILVA, Paula Sofia Valente da - Ketone Bodies as Brain Substrates. Coimbra : [s.n.], 2015. Dissertação de Mestrado em Bioquimica. Since their discovery as a marker for diabetic ketoacidosis, ketone bodies have become known for their therapeutic role as effective agents in refractory epilepsy and a diet specifically designed to increase ketone bodies’ levels in circulation has been often prescribed as treatment. In the classical ketogenic diet, intake of even small additional amounts of car...

  16. Substrate preference of a Geobacillus maltogenic amylase: a kinetic and thermodynamic analysis. (United States)

    Nasrollahi, Samira; Golalizadeh, Leila; Sajedi, Reza H; Taghdir, Majid; Asghari, S Mohsen; Rassa, Mehdi


    The gene encoding a maltogenic amylase (MAase) from a newly isolated strain of thermophilic Geobacillus has been isolated, cloned and expressed. Following purification, biochemical and structural characterization have been performed. The enzyme exhibited maximal activity at a broad temperature range between 55 and 65 °C, clearly higher than that of other dimeric MAses. The optimum pH was 6.0 and catalytic activity increased by of Li(+) and K(+). A noticeable preference was demonstrated for α-, β- and γ-cyclodextrin (CD) compared to polymeric substrates (amylose, amylopectin, glycogen and starch) possibly due to steric interference. The affinity for CD substrates increased in the order of γ-CD>β-CD>α-CD, but k(cat)/K(m) increased as α-CD>β-CD>γ-CD, implying that increased substrate specificities are mainly attribute to kcat. Thermodynamic analysis of the activation process showed that improved activity (decrease in ΔG(#)) is accompanied by increases in activation entropy (ΔS(#)) for aforementioned substrates. Molecular docking on the binding interactions between substrates and active site residues revealed a considerably higher accessible surface area for the active site residues in the presence of α-CD than β-CD, indicating that interactions in the transition state are stronger in the presence of α-CD. This result explains the increased ΔH(#) of the activation process and increased K(m) of the enzyme in the presence of α-CD, compared to that of β-CD. This study, which presents the first detailed comparative analysis on the substrate preference of dimeric MAases for different substrates, may shed some lights into the molecular mechanism of these enzymes.

  17. The broad autism phenotype: findings from an epidemiological survey. (United States)

    Micali, N; Chakrabarti, S; Fombonne, E


    This study aimed to determine if relatives of children with autism and less severe pervasive developmental disorders (PDDs) have higher rates of various components of the broad autistic phenotype. Psychiatric and medical disorders were investigated. Parents of children with PDDs were selected from an epidemiological survey and compared with parents of control children with non-autistic developmental problems. Rates of abnormalities and disorders were compared in relatives of 79 cases and 61 controls. Medical and autoimmune disorders in both groups were endorsed by few relatives. Specific developmental disorders were commoner in parents of controls. Depression and anxiety were significantly more prevalent in mothers of children with PDDs. Significantly more PDD children had at least one first-degree relative with anxiety and one second-degree relative with OCD. PDDs were commoner in first-degree relatives. The implications of the findings for the definition of the broad phenotype of autism are discussed.

  18. Teaching the Broad, Interdisciplinary Impact of Evolution (United States)

    Benson, David; Atlas, Pierre; Haberski, Raymond; Higgs, Jamie; Kiley, Patrick; Maxwell, Michael, Jr.; Mirola, William; Norton, Jamey


    As perhaps the most encompassing idea in biology, evolution has impacted not only science, but other academic disciplines as well. The broad, interdisciplinary impact of evolution was the theme of a course taught at Marian College, Indianapolis, Indiana in 2002, 2004, and 2006. Using a strategy that could be readily adopted at other institutions,…

  19. The GREGOR Broad-Band Imager (United States)

    von der Lühe, O.; Volkmer, R.; Kentischer, T. J.; Geißler, R.


    The design and characteristics of the Broad-Band Imager (BBI) of GREGOR are described. BBI covers the visible spectral range with two cameras simultaneously for a large field and with critical sampling at 390 nm, and it includes a mode for observing the pupil in a Foucault configuration. Samples of first-light observations are shown.

  20. Applications of fibrous substrates containing insolubilized phase change polymers (United States)

    Vigo, Tyrone L.; Bruno, Joseph S.


    Incorporation of polyethylene glycols into fibrous substrates produces several improved functional properties when they are insolubilized by crosslinking with a methylolamide resin or by polyacetal formation by their reaction with glyoxal. The range of molecular weights of polyols that may be insolubilized is broad as are the curing conditions (0.25-10 min at 80-200C). Most representative fiber types and blends (natural and synthetic) and all types of fabric constructions (woven, nonwoven and knit) have been modified by incorporation of the bound polyols. The most novel property is the thermal adaptability of the modified substrates to many climatic conditions. This adaptability is due to the high latent heat of the crosslinked polyols that function as phase change materials, the hydrophilic nature of the crosslinked polymer and its enhanced thermal conductivity. Other enhanced properties imparted to fabrics include flex and flat abrasion, antimicrobial activity, reduced static charge, resistance to oily soils, resiliency, wind resistance and reduced lint loss. Applications commercialized in the U.S. and Japan include sportswear and skiwear. Several examples of electric sets of properties useful for specific end uses are given. In addition, other uses are biomedical horticultural, aerospace, indoor insulation, automotive interiors and components and packaging material.

  1. Broadly Neutralizing Antibodies for HIV Eradication. (United States)

    Stephenson, Kathryn E; Barouch, Dan H


    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  2. Multiple and broad frequency response Gunn diodes (United States)

    Pilgrim, N. J.; Macpherson, R. F.; Khalid, A.; Dunn, G. M.; Cumming, D. R. S.


    Gunn diodes, operating in transit time mode, are usually thought of as incapable of generating power at multiple frequencies or over a broad frequency range. In this paper, we report experimental results showing that these diodes can generate power at several frequencies and, using Monte Carlo simulations of both planar and vertical devices, we offer an explanation of how this unusual behaviour may come into being and suggest possible applications for this novel device.

  3. Substrate stiffness and matrix composition coordinately control the differentiation of liver progenitor cells. (United States)

    Kourouklis, Andreas P; Kaylan, Kerim B; Underhill, Gregory H


    Recent approaches have utilized microfabricated platforms to examine combinations of microenvironmental signals that regulate stem and progenitor cell differentiation. However, the majority of these efforts have focused on the biochemical properties of extracellular matrix (ECM) or soluble factors without simultaneously exploring the biomechanical effects of cell-substrate interactions. To address this need, we combined a high-throughput approach for the analysis of combinatorial ECM cues with substrates of modular stiffness and traction force microscopy. This integrated approach enabled the characterization of cell-generated traction stress and phenotypic expression in response to ECM cues. We investigated the impact of substrate stiffness and ECM composition on the differentiation of bipotential mouse embryonic liver (BMEL) progenitor cells. We observed that hepatocyte differentiation was primarily regulated by ECM composition, and cholangiocyte differentiation was cooperatively influenced by ECM proteins and stiffness properties. In particular, stiffness-mediated cholangiocyte differentiation was observed for cells cultured on fibronectin, while collagen IV promoted differentiation independent of substrate stiffness. We demonstrated the influence of cell contractility and traction stress in early cholangiocyte specification and further uncovered the roles of ERK and ROCK in this differentiation process. Overall, these findings illustrate the involvement of biomechanical signals in liver progenitor differentiation. Further, this approach could enable investigations for a broad range of cell types and ECM proteins, providing an integrated platform for evaluating the combinatorial effects of biochemical and biophysical signals in cell differentiation.

  4. Robust plasmonic substrates

    DEFF Research Database (Denmark)

    Kostiučenko, Oksana; Fiutowski, Jacek; Tamulevicius, Tomas


    substrates is presented, which relies on the coverage of gold nanostructures with diamond-like carbon (DLC) thin films of thicknesses 25, 55 and 105 nm. DLC thin films were grown by direct hydrocarbon ion beam deposition. In order to find the optimum balance between optical and mechanical properties...... and breaking. DLC coating with thicknesses between 25 and 105 nm is found to considerably increase the mechanical strength of the substrates while at the same time ensuring conservation of sufficient field enhancements of the gold plasmonic substrates....

  5. To Broad-Match or Not to Broad-Match : An Auctioneer's Dilemma ?

    CERN Document Server

    Singh, Sudhir Kumar


    We initiate the study of an interesting aspect of sponsored search advertising, namely the consequences of broad match-a feature where an ad of an advertiser can be mapped to a broader range of relevant queries, and not necessarily to the particular keyword(s) that ad is associated with. Starting with a very natural setting for strategies available to the advertisers, and via a careful look through algorithmic and complexity theoretic glasses, we first propose a solution concept called broad match equilibrium(BME) for the game originating from the strategic behavior of advertisers as they try to optimize their budget allocation across various keywords. Next, we consider two broad match scenarios based on factors such as information asymmetry between advertisers and the auctioneer, and the extent of auctioneer's control on the budget splitting. In the first scenario, the advertisers have the full information about broad match and relevant parameters, and can reapportion their own budgets to utilize the extra i...

  6. Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions. (United States)

    Guerin, V; Trinchant, J C; Rigaud, J


    Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r(2) = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Psi(nod)) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C(2)H(2) reduction capacity of mildly water stressed nodules (Psi(nod) = -1.2 megapascals) was possible by increasing the external O(2) partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O(2) diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N(2) fixation activity with glucose than with succinate as an energy-yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O(2), and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N(2) fixation under water restricted conditions.

  7. Crx broadly modulates the pineal transcriptome

    DEFF Research Database (Denmark)

    Rovsing, Louise; Clokie, Samuel; Bustos, Diego M;


    Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. In this study, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use......-regulation of 745 genes (p pineal glands of wild......-type animals; only eight of these were also day/night expressed in the Crx-/- pineal gland. However, in the Crx-/- pineal gland 41 genes exhibited differential night/day expression that was not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also...

  8. Broad spectrum antibiotic compounds and use thereof

    Energy Technology Data Exchange (ETDEWEB)

    Koglin, Alexander; Strieker, Matthias


    The discovery of a non-ribosomal peptide synthetase (NRPS) gene cluster in the genome of Clostridium thermocellum that produces a secondary metabolite that is assembled outside of the host membrane is described. Also described is the identification of homologous NRPS gene clusters from several additional microorganisms. The secondary metabolites produced by the NRPS gene clusters exhibit broad spectrum antibiotic activity. Thus, antibiotic compounds produced by the NRPS gene clusters, and analogs thereof, their use for inhibiting bacterial growth, and methods of making the antibiotic compounds are described.

  9. Broad spectrum microarray for fingerprint-based bacterial species identification

    Directory of Open Access Journals (Sweden)

    Frey Jürg E


    Full Text Available Abstract Background Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. Results A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. Conclusions These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups.

  10. Engineering broadly neutralizing antibodies for HIV prevention and therapy. (United States)

    Hua, Casey K; Ackerman, Margaret E


    A combination of advances spanning from isolation to delivery of potent HIV-specific antibodies has begun to revolutionize understandings of antibody-mediated antiviral activity. As a result, the set of broadly neutralizing and highly protective antibodies has grown in number, diversity, potency, and breadth of viral recognition and neutralization. These antibodies are now being further enhanced by rational engineering of their anti-HIV activities and coupled to cutting edge gene delivery and strategies to optimize their pharmacokinetics and biodistribution. As a result, the prospects for clinical use of HIV-specific antibodies to treat, clear, and prevent HIV infection are gaining momentum. Here we discuss the diverse methods whereby antibodies are being optimized for neutralization potency and breadth, biodistribution, pharmacokinetics, and effector function with the aim of revolutionizing HIV treatment and prevention options.

  11. XMM-Newton and Broad Iron Lines

    CERN Document Server

    Fabian, A C


    Iron line emission is common in the X-ray spectra of accreting black holes. When the line emission is broad or variable then it is likely to originate from close to the black hole. X-ray irradiation of the accretion flow by the power-law X-ray continuum produces the X-ray 'reflection' spectrum which includes the iron line. The shape and variability of the iron lines and reflection can be used as a diagnostic of the radius, velocity and nature of the flow. The inner radius of the dense flow corresponds to the innermost stable circular orbit and thus can be used to determine the spin of the black hole. Studies of broad iron lines and reflection spectra offer much promise for understanding how the inner parts of accretion flows (and outflows) around black holes operate. There remains great potential for XMM-Newton to continue to make significant progress in this work. The need for high quality spectra and thus for long exposure times is paramount.

  12. Crystal structure and biochemical investigations reveal novel mode of substrate selectivity and illuminate substrate inhibition and allostericity in a subfamily of Xaa-Pro dipeptidases. (United States)

    Are, Venkat N; Kumar, Ashwani; Kumar, Saurabh; Goyal, Venuka Durani; Ghosh, Biplab; Bhatnagar, Deepak; Jamdar, Sahayog N; Makde, Ravindra D


    Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

  13. Broad-band acoustic hyperbolic metamaterial

    CERN Document Server

    Shen, Chen; Sui, Ni; Wang, Wenqi; Cummer, Steven A; Jing, Yun


    Acoustic metamaterials (AMMs) are engineered materials, made from subwavelength structures, that exhibit useful or unusual constitutive properties. There has been intense research interest in AMMs since its first realization in 2000 by Liu et al. A number of functionalities and applications have been proposed and achieved using AMMs. Hyperbolic metamaterials are one of the most important types of metamaterials due to their extreme anisotropy and numerous possible applications, including negative refraction, backward waves, spatial filtering, and subwavelength imaging. Although the importance of acoustic hyperbolic metamaterials (AHMMs) as a tool for achieving full control of acoustic waves is substantial, the realization of a broad-band and truly hyperbolic AMM has not been reported so far. Here, we demonstrate the design and experimental characterization of a broadband AHMM that operates between 1.0 kHz and 2.5 kHz.

  14. Magnetohydrodynamic stability of broad line region clouds

    CERN Document Server

    Krause, Martin; Burkert, Andreas


    Hydrodynamic stability has been a longstanding issue for the cloud model of the broad line region in active galactic nuclei. We argue that the clouds may be gravitationally bound to the supermassive black hole. If true, stabilisation by thermal pressure alone becomes even more difficult. We further argue that if magnetic fields should be present in such clouds at a level that could affect the stability properties, they need to be strong enough to compete with the radiation pressure on the cloud. This would imply magnetic field values of a few Gauss for a sample of Active Galactic Nuclei we draw from the literature. We then investigate the effect of several magnetic configurations on cloud stability in axi-symmetric magnetohydrodynamic simulations. For a purely azimuthal magnetic field which provides the dominant pressure support, the cloud first gets compressed by the opposing radiative and gravitational forces. The pressure inside the cloud then increases, and it expands vertically. Kelvin-Helmholtz and colu...

  15. Spectrophotometry of six broad absorption line QSOs (United States)

    Junkkarinen, Vesa T.; Burbidge, E. Margaret; Smith, Harding E.


    Spectrophotometric observations of six broad absorption-line QSOs (BALQSOs) are presented. The continua and emission lines are compared with those in the spectra of QSOs without BALs. A statistically significant difference is found in the emission-line intensity ratio for (N V 1240-A)/(C IV 1549-A). The median value of (N V)/(C IV) for the BALQSOs is two to three times the median for QSOs without BALs. The absorption features of the BALQSOs are described, and the column densities and limits on the ionization structure of the BAL region are discussed. If the dominant ionization mechanism is photoionization, then it is likely that either the ionizing spectrum is steep or the abundances are considerably different from solar. Collisional ionization may be a significant factor, but it cannot totally dominate the ionization rate.

  16. PREFACE: Cell-substrate interactions Cell-substrate interactions (United States)

    Gardel, Margaret; Schwarz, Ulrich


    not on the amount of ligand for adhesion receptors, but on its spatial distribution [1]. New protocols for the preparation of soft elastic substrates were essential to show that adhesion structures and cytoskeleton of adherent cells strongly adapt to substrate stiffness [2], with dramatic effects for cellular decision making. For example, it has been shown recently that differentiation of mesenchymal stem cells is strongly influenced by substrate stiffness [3]. Thus, physical factors appear to be equally important as biochemical ones in determining the cellular response to its substrate [4]. The introduction of novel physical techniques not only opened up completely new perspectives regarding biological function, it also introduced a new quantitative element into this field. For example, the availability of soft elastic substrates with controlled stiffness allows us to reconstruct cellular traction forces and to correlate them with other cellular features. This development enables modeling approaches to work in close contact with experimental data, thus opening up the perspective that the field of cell-substrate interactions will become a quantitative and predictive science in the future. Because physical research into cell-substrate interactions has become one of the fastest growing research areas in cellular biophysics and materials science, we believe that it is very timely that this special issue gathers some of the on-going research effort in this field. In contrast to the non-living world, cellular systems usually interact with their environment through specific adhesion, mainly based on adhesion receptors from the integrin family. During recent years, force spectroscopy has emerged as one of the main methods to study the physics of specific adhesion. In this special issue, single cell force spectroscopy is used by Boettiger and Wehrle-Haller to characterize the strength of cell-matrix adhesion and how it is modulated by the glycocalyx [5], while Chirasatitsin

  17. Chemometrics approach to substrate development, case: semisyntetic cheese

    DEFF Research Database (Denmark)

    Nielsen, Per Væggemose; Hansen, Birgitte Vedel


    In several cases a well defined, robust and easy reproducible substrate that meets specific requirements is needed. This is the case in studies of fungal growth and metabolism on specific products as affected by environmental conditions or processing factors, or isolation of product specific fungi...... from food production facilities.The Chemometrics approach to substrate development is illustrated by the development of a semisyntetic cheese substrate. Growth, colour formation and mycotoxin production of 6 cheese related fungi were studied on 9 types of natural cheeses and 24 synthetic cheese...... substrates and compared using principal component analysis (PCA). The synthetic cheese substrates contained various amounts of Ca, K, Mg, Na, P, Fe, Cu, Zn, lactate, lactose and casein. In this manner a robust, well-defined and easy prepared laboratory cheese substrate was developed for Penicillium commune...

  18. Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3

    DEFF Research Database (Denmark)

    Wandall, H H; Hassan, H; Mirgorodskaya, E;


    Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified...

  19. A substrate specificity-determining unit of three Lin12-Notch repeat modules is formed in trans within the pappalysin-1 dimer and requires a sequence stretch C-terminal to the third module

    DEFF Research Database (Denmark)

    Weyer, Kathrin; Boldt, Henning B; Poulsen, Christine B;


    Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP...

  20. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate

    DEFF Research Database (Denmark)

    Grønborg, Mads; Kristiansen, Troels Zakarias; Stensballe, Allan;


    proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine...

  1. Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV)

    DEFF Research Database (Denmark)

    Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall


    for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using...

  2. Seven broad absorption line quasars with excess broad band absorption near 2250

    CERN Document Server

    Zhang, Shaohua; Jiang, Peng; Zhou, Hongyan; Ma, Jingzhe; Brandt, W N; York, Donald G; Noterdaeme, P; Schneider, Donald P


    We report the discovery of excess broad band absorption near 2250 A (EBBA) in the spectra of seven broad absorption line (BAL) quasars. By comparing with the statistical results from the control quasar sample, the significance for the detections are all above the > 4{\\sigma} level, with five above > 5{\\sigma}. The detections have also been verified by several other independent methods. The EBBAs present broader and weaker bumps at smaller wavenumbers than the Milky Way, and similar to the Large Magellanic Cloud. The EBBA bump may be related to the 2175 A bump seen in the Local Group and may be a counterpart of the 2175 A bump under different conditions in the early Universe. Furthermore, five objects in this sample show low-ionization broad absorption lines (LoBALs), such as Mg II and Al III, in addition to the high-ionization broad absorption lines (HiBALs) of C IV and Si IV. The fraction of LoBALs in our sample, ~70%, is surprisingly high compared to that of general BAL quasars, ~10%. Although the origin of...

  3. Multivalent dendritic molecules as broad spectrum bacteria agglutination agents. (United States)

    Xiao, Shuzhang; Abu-Esba, Lica; Turkyilmaz, Serhan; White, Alexander G; Smith, Bradley D


    This study reports the first set of synthetic molecules that act as broad spectrum agglutination agents and thus are complementary to the specific targeting of antibodies. The molecules have dendritic architecture and contain multiple copies of zinc(II)-dipicolylamine (ZnDPA) units that have selective affinity for the bacterial cell envelope. A series of molecular structures were evaluated, with the number of appended ZnDPA units ranging from four to thirty-two. Agglutination assays showed that the multivalent probes rapidly cross-linked ten different strains of bacteria, regardless of Gram-type and cell morphology. Fluorescence microscopy studies using probes with four ZnDPA units indicated a high selectivity for bacteria agglutination in the presence of mammalian cells and no measurable effect on the health of the cells. The high bacterial selectivity was confirmed by conducting in vivo optical imaging studies of a mouse leg infection model. The results suggest that multivalent ZnDPA molecular probes with dendritic structures have great promise as selective, broad spectrum bacterial agglutination agents for infection imaging and theranostic applications.

  4. Electrophoretic deposition and characterization of nanocomposites and nanoparticles on magnesium substrates (United States)

    Tian, Qiaomu; Liu, Huinan


    This study introduces a triphasic design of biodegradable materials composed of nanophase hydroxyapatite (nHA), poly(lactic-co-glycolic acid) (PLGA), and magnesium (Mg) substrates for musculoskeletal implant applications. Specifically, nHA_PLGA composites and nHA nanoparticles were synthesized, deposited on three-dimensional Mg substrates using electrophoretic deposition (EPD), and characterized. The three components involved, that is, nHA, PLGA, and Mg are all biodegradable in the human body, thus promising for biodegradable implant and device applications. Mg and its alloys are attractive for musculoskeletal implant applications due to their comparable modulus and strength to cortical bone. Controlling the interface of Mg with the biological environment, however, is the key challenge that currently limits this biodegradable metal for broad applications in medical implants. This article particularly focuses on creating nanostructured interface between the biodegradable Mg and surrounding tissue for the dual purposes of (1) mediating the degradation of the Mg-based substrates and (2) potentially enhancing osteointegration. Nanophase hydroxyapatite (nHA) is an excellent candidate as a coating material due to its osteoconductivity, while the polymer phase promotes interfacial adhesion between the nHA and Mg. Moreover, the degradation products of PLGA and Mg neutralize each other. Surface characterization showed successful deposition of nHA_PLGA composite microspheres and nHA nanoparticles on Mg substrates using EPD. Mg substrates coated with nHA_PLGA composites showed greater adhesion strength when compared with nHA coating, and slower corrosion rate than nHA coated Mg and non-coated Mg. The triphasic composites of nHA, PLGA and Mg are promising as the next-generation biodegradable materials for medical applications.

  5. Photoionisation modelling of the broad line region (United States)

    King, Anthea


    Two of the most fundamental questions regarding the broad line region (BLR) are "what is its structure?" and "how is it moving?" Baldwin et al. (1995) showed that by summing over an ensemble of clouds at differing densities and distances from the ionising source we can easily and naturally produce a spectrum similar to what is observed for AGN. This approach is called the `locally optimally emitting clouds' (LOC) model. This approach can also explain the well-observed stratification of emission lines in the BLR (e.g. Clavel et al. 1991, Peterson et al. 1991, Kollatschny et al. 2001) and `breathing' of BLR with changes in the continuum luminosity (Netzer & Mor 1990, Peterson et al. 2014) and is therefore a generally accepted model of the BLR. However, LOC predictions require some assumptions to be made about the distribution of the clouds within the BLR. By comparing photoionization predictions, for a distribution of cloud properties, with observed spectra we can infer something about the structure of the BLR and distribution of clouds. I use existing reverberation mapping data to constrain the structure of the BLR by observing how individual line strengths and ratios of different lines change in high and low luminosity states. I will present my initial constraints and discuss the challenges associated with the method.

  6. Rufinamide: a novel broad-spectrum antiepileptic drug. (United States)

    Wheless, James W; Vazquez, Blanca


    The last 20 years have witnessed a tremendous explosion in the number of antiepileptic drugs (AEDs) as well as the introduction of AEDS developed for specific epilepsy syndromes. The study of the efficacy and side effect profile of AEDs for unique epilepsy syndromes has allowed neurologists to utilize evidence-based medicine when treating patients. In late 2008, the Food and Drug Administration approved rufinamide for adjunctive use in the treatment of seizures associated with Lennox-Gastaut syndrome. This unique chemical compound is also the first new AED to reach the market in the United States having a pediatric indication prior to approval for adults. Rufinamide appears to have a broad spectrum of efficacy, is well tolerated, and may be rapidly initiated--properties that will likely extend its use outside of Lennox-Gastaut syndrome.

  7. Ultra-Broad-Band Optical Parametric Amplifier or Oscillator (United States)

    Strekalov, Dmitry; Matsko, Andrey; Savchenkov, Anatolly; Maleki, Lute


    A concept for an ultra-broad-band optical parametric amplifier or oscillator has emerged as a by-product of a theoretical study in fundamental quantum optics. The study was originally intended to address the question of whether the two-photon temporal correlation function of light [in particular, light produced by spontaneous parametric down conversion (SPDC)] can be considerably narrower than the inverse of the spectral width (bandwidth) of the light. The answer to the question was found to be negative. More specifically, on the basis of the universal integral relations between the quantum two-photon temporal correlation and the classical spectrum of light, it was found that the lower limit of two-photon correlation time is set approximately by the inverse of the bandwidth. The mathematical solution for the minimum two-photon correlation time also provides the minimum relative frequency dispersion of the down-converted light components; in turn, the minimum relative frequency dispersion translates to the maximum bandwidth, which is important for the design of an ultra-broad-band optical parametric oscillator or amplifier. In the study, results of an analysis of the general integral relations were applied in the case of an optically nonlinear, frequency-dispersive crystal in which SPDC produces collinear photons. Equations were found for the crystal orientation and pump wavelength, specific for each parametric-down-converting crystal, that eliminate the relative frequency dispersion of collinear degenerate (equal-frequency) signal and idler components up to the fourth order in the frequency-detuning parameter

  8. Broad spectrum anthelmintic potential of Cassia plants

    Institute of Scientific and Technical Information of China (English)

    Suman Kundu; Saptarshi Roy; Larisha Mawkhleing Lyndem


    Objective: To study the in vitro anthelmintic efficacy of Cassia alata (C. alata), Cassia(C. angustifolia) and Cassia occidentalis (C. occidentalis). angustifolia Methods: Crude ethanol extract from leaves of the three plants were prepared in rotary evaporator and different concentrations (10, 20 and 40 mg/mL) of leaf extracts were used for treatment on different representatives of helminthes (Heterakis gallinarum, Raillietina tetragona and Catatropis sp.) from domestic fowl (Gallus gallus domesticus). Loss of motility and death were monitored frequently.Results: C. alata showed early paralysis in all worms treated followed by C. angustifolia. C. occidentalis in combination with C. alata together caused early paralysis in all treated worms than the combination of C. alata with C. angustfolia. While Heterakis gallinarum in control survived for (81.33±2.07) h, treated worms lost their motility at (5.71±0.10) h, (6.60±0.86) h and (13.95±0.43) h with C. angustifolia, C. alata and C. occidentalis respectively at a concentration of 40 mg/mL which showed better efficacy than albendazole. Catatropis sp. survival period was (26.49±1.38) h in control, but with plant treatment, it lost its motility in just (0.57±0.08) h, (1.00±0.12) h and (1.47±0.40) h at 40 mg/mL concentration of C. alata, C. angustifolia and C. occidentalis respectively.Raillietina tetragona on the other hand became paralysed at (1.68±0.27) h, (2.95±0.29) h and (4.13±0.31) h with above concentrations treated with three plants respectively, however in control it survived up to (81.93±4.71) h.Conclusions:This present study indicated broad spectrum vermifugal activity of all plants tested.

  9. Arctic Change Information for a Broad Audience (United States)

    Soreide, N. N.; Overland, J. E.; Calder, J.


    Demonstrable environmental changes have occurred in the Arctic over the past three decades. NOAA's Arctic Theme Page is a rich resource web site focused on high latitude studies and the Arctic, with links to widely distributed data and information focused on the Arctic. Included is a collection of essays on relevant topics by experts in Arctic research. The website has proven useful to a wide audience, including scientists, students, teachers, decision makers and the general public, as indicated through recognition by USA Today, Science magazine, etc. ( Working jointly with NSF and the University of Washington's Polar Science Center as part of the Study of Environmental Arctic Change (SEARCH) program, NOAA has developed a website for access to pan-Arctic time series spanning diverse data types including climate indices, atmospheric, oceanic, sea ice, terrestrial, biological and fisheries. Modest analysis functions and more detailed analysis results are provided. ( This paper will describe development of an Artic Change Detection status website to provide a direct and comprehensive view of previous and ongoing change in the Arctic for a broad climate community. For example, composite metrics are developed using principal component analysis based on 86 multivariate pan-Arctic time series for seven data types. Two of these metrics can be interpreted as a regime change/trend component and an interdecadal component. Changes can also be visually observed through tracking of 28 separate biophysical indicators. Results will be presented in the form of a web site with relevant, easily understood, value-added knowledge backed by peer review from Arctic scientists and scientific journals.

  10. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  11. Structure and energetics of helium films on alkali substrates (United States)

    Boninsegni, Massimo; Szybisz, Leszek


    Low-temperature adsorption of He4 films on Alkali metal substrates is investigated theoretically by means of ground-state quantum Monte Carlo simulations. The most accurate potentials currently available are utilized to model the interaction of He4 atoms with the substrate. Continuous growth of film thickness as a function of chemical potential is observed on Li, Na, and K substrates. A superfluid monolayer forms on a Li substrate; on Na and K, thermodynamically stable films are a few layers thick. The uncertainties of the calculation and in the potentials, preclude a definitive conclusion on the existence of a stable He4 film on Rb. A comparison of the results of this calculation with those obtained using the Orsay-Trento density functional shows broad quantitative agreement.

  12. Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase. (United States)

    Maksimov, Mikhail O; Koos, Joseph D; Zong, Chuhan; Lisko, Bozhena; Link, A James


    Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.

  13. Broad-Spectrum Solution-Processed Photovoltaics (United States)

    Ip, Alexander Halley

    High global demand for energy coupled with dwindling fossil fuel supply has driven the development of sustainable energy sources such as solar photovoltaics. Emerging solar technologies aim for low-cost, solution-processable materials which would allow wide deployment. Colloidal quantum dots (CQDs) are such a materials system which exhibits the ability to absorb across the entire solar spectrum, including in the infrared where many technologies cannot harvest photons. However, due to their nanocrystalline nature, CQDs are susceptible to surface-associated electronic traps which greatly inhibit performance. In this thesis, surface engineering of CQDs is presented through a combined ligand approach which improves the passivation of surface trap states. A metal halide treatment is found to passivate quantum dot surfaces in solution, while bifunctional organic ligands produce a dense film in solid state. This approach reduced midgap trap states fivefold compared with conventional passivation strategies and led to solar cells with a record certified 7.0% power conversion efficiency. The effect of this process on the electronic structure is studied through photoelectron spectroscopy. It is found that while the halide provides deep trap passivation, the nature of the metal cation on the CQD surface affects the density of band tail states. This effect is explored further through a wide survey of materials, and it is found that the coordination ability of the metal cation is responsible for the suppression of shallow traps. With this understanding of CQD surface passivation, broad spectral usage is then explored through a study of visible-absorbing organolead halide perovskite materials as well as narrow-bandgap CQD solar cells. Control over growth conditions and modification of electrode interfaces resulted in efficient perovskite devices with effective usages of visible photons. For infrared-absorbing CQDs, it is found that, in addition to providing surface trap

  14. Kinetics Modelling of the Biodegradation of Benzene, Toluene and Phenol as Single Substrate and Mixed Substrate by Using Pseudomonas putida


    Mathur, A K; Majumder, C. B.


    In the present work, kinetics of the biodegradation of benzene, toluene and phenol by using a pure culture of Pseudomonas putida (MTCC 1194) was determined by measuring the specific growth rate and degradation rate with substrate concentration as a function of time in a batch reactor. In general, the degradation rate of benzene, toluene and phenol increased with the increase in the initial substrate concentration and then decreased after reaching a maximum, showing substrate inhibition kineti...

  15. Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

    NARCIS (Netherlands)

    Biesebeke, R. te; Boussier, A.; Biezen, N. van; Hondel, C.A.M.J.J. van den; Punt, P.J.


    Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media

  16. Substrate Material for Holographic Emulsions Utilizing Fluorinated Polyimide Film (United States)

    Gierow, Paul A. (Inventor); Clayton, William R. (Inventor); St.Clair, Anne K. (Inventor)


    A new holographic substrate utilizing flexible. optically transparent fluorinated polyimides. Said substrates have 0 extremely low birefringence which results in a high signal to noise ratio in subsequent holograms. Specific examples of said fluorinated polyimides include 6FDA+APB and 6FDA+4BDAF.

  17. Substrate recognition by the 2-hydroxycarboxylate transport proteins

    NARCIS (Netherlands)

    Bandell, Michael


    The object of the research described in this thesis was to identify substrate-protein interactions that provide affinity and specificity for CitPLEME from Lc.mesenteroides and MlePLALA form L.lactis. The ability of these transporters to transport substrates that differ in structure and charge implie

  18. Substrate recognition by ribonucleoprotein ribonuclease MRP. (United States)

    Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S


    The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

  19. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface


    Huang, Jinghe; Kang, Byong H; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald


    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in 1 ). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50

  20. Dewetting on microstructured substrates (United States)

    Kim, Taehong; Kim, Wonjung


    A thin liquid film has an equilibrium thickness in such a way as to minimize the free energy. When a liquid film thickness is out of its equilibrium, the film seeks its equilibrium state, resulting in dynamics of liquid film, which are referred to as wetting and dewetting, depending on the flow direction. We here present a combined experimental and theoretical investigation of dewetting on a substrate with parallel microstructures. Our experiments show that residue may remain on the substrate after dewetting, and residue morphologies can be classified into three modes. Based on our experimental observations, we elucidate how the modes depend on the pattern morphology and contact angle, and develop a model for the contact line motion. Our results provide a basis for controlling the thickness film, which is important for many practical applications such as oil recovery, detergency, lithography, and cleaning. This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIP) (No.2015R1A2A2A04006181).

  1. Reactive Oxygen Species and the Aging Eye: Specific Role of Metabolically Active Mitochondria in Maintaining Lens Function and in the Initiation of the Oxidation-Induced Maturity Onset Cataract--A Novel Platform of Mitochondria-Targeted Antioxidants With Broad Therapeutic Potential for Redox Regulation and Detoxification of Oxidants in Eye Diseases. (United States)

    Babizhayev, Mark A; Yegorov, Yegor E


    -targeted rechargeable antioxidant (either MitoVit E, Mito Q, or SkQs) as a potent medicine to treat ocular diseases. Such specificity is explained by the fact that developed compositions might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo and outside mitochondria in the cellular and tissue structures of the lens and eye compartments. Mitochondrial targeting of compounds with universal types of antioxidant activity represents a promising approach for treating a number of ROS-related ocular diseases of the aging eye and can be implicated in the management of cataracts.

  2. Maintainable substrate carrier for electroplating (United States)

    Chen, Chen-An [Milpitas, CA; Abas, Emmanuel Chua [Laguna, PH; Divino, Edmundo Anida [Cavite, PH; Ermita, Jake Randal G [Laguna, PH; Capulong, Jose Francisco S [Laguna, PH; Castillo, Arnold Villamor [Batangas, PH; Ma,; Xiaobing, Diana [Saratoga, CA


    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The carrier includes a non-conductive carrier body on which the substrates are placed and conductive lines embedded within the carrier body. A plurality of conductive clip attachment parts are attached in a permanent manner to the conductive lines embedded within the carrier body. A plurality of contact clips are attached in a removable manner to the clip attachment parts. The contact clips hold the substrates in place and conductively connecting the substrates with the conductive lines. Other embodiments, aspects and features are also disclosed.

  3. Maintainable substrate carrier for electroplating

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chen-An; Abas, Emmanuel Chua; Divino, Edmundo Anida; Ermita, Jake Randal G.; Capulong, Jose Francisco S.; Castillo, Arnold Villamor; Ma, Diana Xiaobing


    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The carrier includes a non-conductive carrier body on which the substrates are placed and conductive lines embedded within the carrier body. A plurality of conductive clip attachment parts are attached in a permanent manner to the conductive lines embedded within the carrier body. A plurality of contact clips are attached in a removable manner to the clip attachment parts. The contact clips hold the substrates in place and conductively connecting the substrates with the conductive lines. Other embodiments, aspects and features are also disclosed.

  4. On the Operational Meaning of Broad-Spectrum Philosophy Methodology

    Directory of Open Access Journals (Sweden)

    Miao Yi


    Full Text Available This study analyze the Broad-spectrum Philosophy Methodology on the operational meaning, and discusses three relatively primary methods of Broad-spectrum Philosophy Methodologies. The methodology of Broad-spectrum philosophy cannot pursuit of accuracy of numerical sense, but it pursuits of the exact nature of the research questions, the accuracy of the sense of set theory and sums up the experience which we solve problems in real life and make it up to the generalized quantization and procedural level and thus the methodology of Broad-spectrum philosophy has a general guide and it is a broad operations research. We gets the results that along with deep exploration and development of Broad-spectrum Philosophy Methodology, its function as generalized operation research can be more powerful.

  5. Experimental analysis of green roof substrate detention characteristics. (United States)

    Yio, Marcus H N; Stovin, Virginia; Werdin, Jörg; Vesuviano, Gianni


    Green roofs may make an important contribution to urban stormwater management. Rainfall-runoff models are required to evaluate green roof responses to specific rainfall inputs. The roof's hydrological response is a function of its configuration, with the substrate - or growing media - providing both retention and detention of rainfall. The objective of the research described here is to quantify the detention effects due to green roof substrates, and to propose a suitable hydrological modelling approach. Laboratory results from experimental detention tests on green roof substrates are presented. It is shown that detention increases with substrate depth and as a result of increasing substrate organic content. Model structures based on reservoir routing are evaluated, and it is found that a one-parameter reservoir routing model coupled with a parameter that describes the delay to start of runoff best fits the observed data. Preliminary findings support the hypothesis that the reservoir routing parameter values can be defined from the substrate's physical characteristics.

  6. Cross-reactive broadly neutralizing antibodies: timing is everything


    Euler, Zelda; Schuitemaker, Hanneke


    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  7. Cross-reactive broadly neutralizing antibodies: timing is everything. (United States)

    Euler, Zelda; Schuitemaker, Hanneke


    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  8. Multifunctional epitaxial systems on silicon substrates (United States)

    Singamaneni, Srinivasa Rao; Prater, John Thomas; Narayan, Jagdish


    Multifunctional heterostructures can exhibit a wide range of functional properties, including colossal magneto-resistance, magnetocaloric, and multiferroic behavior, and can display interesting physical phenomena including spin and charge ordering and strong spin-orbit coupling. However, putting this functionality to work remains a challenge. To date, most of the work reported in the literature has dealt with heterostructures deposited onto closely lattice matched insulating substrates such as DyScO3, SrTiO3 (STO), or STO buffered Si(100) using concepts of lattice matching epitaxy (LME). However, strain in heterostructures grown by LME is typically not fully relaxed and the layers contain detrimental defects such as threading dislocations that can significantly degrade the physical properties of the films and adversely affect the device characteristics. In addition, most of the substrates are incompatible with existing CMOS-based technology, where Si (100) substrates dominate. This review discusses recent advances in the integration of multifunctional oxide and non-oxide materials onto silicon substrates. An alternative thin film growth approach, called "domain matching epitaxy," is presented which identifies approaches for minimizing lattice strain and unwanted defects in large misfit systems (7%-25% and higher). This approach broadly allows for the integration of multifunctional materials onto silicon substrates, such that sensing, computation, and response functions can be combined to produce next generation "smart" devices. In general, pulsed laser deposition has been used to epitaxially grow these materials, although the concepts developed here can be extended to other deposition techniques, as well. It will be shown that TiN and yttria-stabilized zirconia template layers provide promising platforms for the integration of new functionality into silicon-based computer chips. This review paper reports on a number of thin-film heterostructure systems that span a

  9. A Low Cost High Specific Stiffness Mirror Substrate Project (United States)

    National Aeronautics and Space Administration — The primary purpose of this proposal is to develop and demonstrate a new technology for manufacturing an ultra-low-cost precision optical telescope mirror which can...

  10. Substrate specificity of hybrid modules from peptide synthetases

    NARCIS (Netherlands)

    Elsner, A; Engert, H; Saenger, W; Hamoen, L; Venema, G; Bernhard, F


    Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine activating module of surfactin synthetase were constructed by combining the t

  11. Photocatalytic water treatment: substrate-specific activity of titanium dioxide

    NARCIS (Netherlands)

    Sobral Romao, Joana Isabel


    In this thesis, heterogeneous photocatalysis is presented as a promising technology for water purification. Photocatalysis is revisited from an experimental and engineering point of view, comprising some theoretical fundamentals towards the commercialization of domestic appliances for water purifica

  12. Two nucleoside transporters in Lactococcus lactis with different substrate specificities

    DEFF Research Database (Denmark)

    Martinussen, Jan; Sørensen, Claus; Jendresen, Christian Bille


    In an alternative to biosynthesis of nucleotides, most organisms are capable of exploiting exogenous nucleotide sources. In order to do so, the nucleotide precursors must pass the membrane, which requires the presence of transporters. Normally, phosphorylated compounds are not subject to transport...

  13. Inclination of Broad Line Region in Narrow Line and Broad Line Seyfert 1 Galaxies

    Institute of Scientific and Technical Information of China (English)


    The sizes of the Broad Line Region (BLR) of some Seyfert 1 galax-ies and nearby quasars can be determined by the reverberation mapping method.Combining with the observed FWHM of Hβ emission line and assuming that themotion of BLR clouds is virialized, the black hole masses of these objects have beenestimated. However, this method strongly depends on the poorly-understood geom-etry and inclination of the BLR. On the other hand, a tight correlation between theblack hole mass and the bulge velocity dispersion was recently found for both activeand nearby inactive galaxies. This may provide another method, independent of theBLR geometry, for estimating the black hole mass. Using this method for estimatingthe black hole mass and combining with the measured BLR size and FWHM of Hβemission line, we derived the BLR inclination angles for 20 Seyfert I galaxies underthe assumption that the BLR is disk-like. The derived inclination angles agree wellwith those derived previously by fitting the UV continuum and Hβ emission lineprofiles. Adopting a relation between the FWHMs of [OⅢ]λ5007 forbidden line andthe stellar velocity dispersion, we also estimated the BLR inclinations for 50 nar-row line Seyfert 1 galaxies (NLSls). We found that the inclinations of broad LineSeyfert 1 galaxies (BLS1s) are systematically greater than those of NLS1s, whichseldom exceed 30. This may be an important factor that leads to the differencesbetween NLS1s and BLS1s if the BLR of NLS1s is really disk-like.

  14. Nuclear RNA surveillance: role of TRAMP in controlling exosome specificity. (United States)

    Schmidt, Karyn; Butler, J Scott


    The advent of high-throughput sequencing technologies has revealed that pervasive transcription generates RNAs from nearly all regions of eukaryotic genomes. Normally, these transcripts undergo rapid degradation by a nuclear RNA surveillance system primarily featuring the RNA exosome. This multimeric protein complex plays a critical role in the efficient turnover and processing of a vast array of RNAs in the nucleus. Despite its initial discovery over a decade ago, important questions remain concerning the mechanisms that recruit and activate the nuclear exosome. Specificity and modulation of exosome activity requires additional protein cofactors, including the conserved TRAMP polyadenylation complex. Recent studies suggest that helicase and RNA-binding subunits of TRAMP direct RNA substrates for polyadenylation, which enhances their degradation by Dis3/Rrp44 and Rrp6, the two exosome-associated ribonucleases. These findings indicate that the exosome and TRAMP have evolved highly flexible functions that allow recognition of a wide range of RNA substrates. This flexibility provides the nuclear RNA surveillance system with the ability to regulate the levels of a broad range of coding and noncoding RNAs, which results in profound effects on gene expression, cellular development, gene silencing, and heterochromatin formation. This review summarizes recent findings on the nuclear RNA surveillance complexes, and speculates upon possible mechanisms for TRAMP-mediated substrate recognition and exosome activation.

  15. HAMLET - A protein-lipid complex with broad tumoricidal activity. (United States)

    Ho, James C S; Nadeem, Aftab; Svanborg, Catharina


    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

  16. A Broad Set of Chromatin Factors Influences Splicing (United States)

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian


    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  17. Recognition determinants of broadly neutralizing human antibodies against dengue viruses. (United States)

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A


    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  18. Assessing Eli Broad's Assault on Public School System Leadership (United States)

    English, Fenwick W.; Crowder, Zan


    Eli Broad's approach to reforming urban public education does not recognize his own self-interest in promoting changes within such educational systems, a classic problem of misrecognition. The Broad agenda is an assault on the notion of the mission of public education as a service instead of a for-profit enterprise concerned with making money for…

  19. Social Cognition, Social Skill, and the Broad Autism Phenotype (United States)

    Sasson, Noah J.; Nowlin, Rachel B.; Pinkham, Amy E.


    Social-cognitive deficits differentiate parents with the "broad autism phenotype" from non-broad autism phenotype parents more robustly than other neuropsychological features of autism, suggesting that this domain may be particularly informative for identifying genetic and brain processes associated with the phenotype. The current study…

  20. Broadly protective influenza vaccines: Redirecting the antibody response through adjuvation

    NARCIS (Netherlands)

    Cox, F.


    Influenza virus infections are responsible for significant morbidity worldwide and current vaccines have limited coverage, therefore it remains a high priority to develop broadly protective vaccines. With the discovery of broadly neutralizing antibodies (bnAbs) against influenza these vaccines becam

  1. Boot Camp for Education CEOs: The Broad Foundation Superintendents Academy (United States)

    Jehlen, Alain


    The Broad Foundation Superintendents Academy is the most prominent and most controversial training institute for school chiefs. The Academy is the flagship program of the Eli and Edythe Broad Foundation, the smallest of a triumvirate of corporate foundations that are at the heart of the billionaire campaign to remake public education in the image…

  2. Flexible and tunable silicon photonic circuits on plastic substrates

    CERN Document Server

    Chen, Yu; Li, Mo


    Flexible microelectronics has shown tremendous promise in a broad spectrum of applications, especially those that cannot be addressed by conventional microelectronics in rigid materials and constructions1-3. These unconventional yet important applications range from flexible consumer electronics to conformal sensor arrays and biomedical devices. A recent successful paradigm shift in implementing flexible electronics is to physically transfer and bond highly integrated devices made in high-quality, crystalline semiconductor materials on to plastic materials4-8. Here we demonstrate a flexible form of silicon photonics on plastic substrates using the transfer-and-bond fabrication method. Photonic circuits including interferometers and resonators have been transferred onto flexible plastic substrates with preserved functionalities and performance. By mechanically deforming the flexible substrates, the optical characteristics of the devices can be tuned reversibly over a remarkably large range. The demonstration o...

  3. [Biogas production from cellulose-containing substrates: a review]. (United States)

    Tsavkelova, E A; Netrusov, A I


    Anaerobic microbial conversion of organic substrates to various biofuels is one of the alternative energy sources attracting the greatest attention of scientists. The advantages of biogas production over other technologies are the ability of methanogenic communities to degrade a broad range of substrates and concomitant benefits: neutralization of organic waste, reduction of greenhouse gas emission, and fertilizer production. Cellulose-containing materials are a good substrate, but their full-scale utilization encounters a number of problems, including improvement of the quality and amount ofbiogas produced and maintenance of the stability and high efficiency of microbial communities. We review data on microorganisms that form methanogenic cellulolytic communities, enzyme complexes of anaerobes essential for cellulose fiber degradation, and feedstock pretreatment, as biodegradation is hindered in the presence of lignin. Methods for improving biogas production by optimization of microbial growth conditions are considered on the examples of biogas formation from various types of plant and paper materials: writing paper and cardboard.

  4. Design of an Ultra Broad Band YIG-Tuned FET Oscillator Operating within -45~+65℃

    Institute of Scientific and Technical Information of China (English)


    In this paper, the design of an ultra broad band YIG-Tuned FET oscillator is briefly presented. These performances of the superior linearity, the pure spectrum and the ultra broad band tuned oscillation have been realized by a common source circuit topology and a dual coupling YIG resonator. Using a BeO substrate and a buffering amplification stage made up of monolithic MIC traveling wave amplifier, the RF output power has been obtained above 13 dBm in the range from 4 to 16.5 GHz; By means of YIG heater and the temperature compensation of the magnetic circuit, the broad temperature operation performance of YTO has been realized within -45~+65℃.

  5. Current models broadly neglect specific needs of biodiversity conservation in protected areas under climate change

    Directory of Open Access Journals (Sweden)

    Moloney Kirk A


    Full Text Available Abstract Background Protected areas are the most common and important instrument for the conservation of biological diversity and are called for under the United Nations' Convention on Biological Diversity. Growing human population densities, intensified land-use, invasive species and increasing habitat fragmentation threaten ecosystems worldwide and protected areas are often the only refuge for endangered species. Climate change is posing an additional threat that may also impact ecosystems currently under protection. Therefore, it is of crucial importance to include the potential impact of climate change when designing future nature conservation strategies and implementing protected area management. This approach would go beyond reactive crisis management and, by necessity, would include anticipatory risk assessments. One avenue for doing so is being provided by simulation models that take advantage of the increase in computing capacity and performance that has occurred over the last two decades. Here we review the literature to determine the state-of-the-art in modeling terrestrial protected areas under climate change, with the aim of evaluating and detecting trends and gaps in the current approaches being employed, as well as to provide a useful overview and guidelines for future research. Results Most studies apply statistical, bioclimatic envelope models and focus primarily on plant species as compared to other taxa. Very few studies utilize a mechanistic, process-based approach and none examine biotic interactions like predation and competition. Important factors like land-use, habitat fragmentation, invasion and dispersal are rarely incorporated, restricting the informative value of the resulting predictions considerably. Conclusion The general impression that emerges is that biodiversity conservation in protected areas could benefit from the application of modern modeling approaches to a greater extent than is currently reflected in the scientific literature. It is particularly true that existing models have been underutilized in testing different management options under climate change. Based on these findings we suggest a strategic framework for more effectively incorporating the impact of climate change in models exploring the effectiveness of protected areas.

  6. Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

    NARCIS (Netherlands)

    Eerde, André van; Hamiaux, Cyril; Pérez, Javier; Parsot, Claude; Dijkstra, Bauke W.


    Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 f

  7. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma. (United States)

    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L


    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

  8. Compound-Specific Isotope Analysis of Nitroaromatic Contaminant Transformations by Nitroarene Dioxygenases (United States)

    Pati, Sarah G.; Kohler, Hans-Peter E.; Hofstetter, Thomas B.


    Dioxygenation is an important biochemical reaction that often initiates the mineralization of recalcitrant organic contaminants such as nitroaromatic explosives, chlorinated benzenes, and polycyclic aromatic hydrocarbons. However, to assess the extent of dioxygenation in contaminated environments is difficult because of competing transformation processes and further reactions of the dioxygenation products. Compound-specific isotope analysis (CSIA) offers a new approach to reliably quantify biodegradation initiated by dioxygenation based on changes in stable isotope ratios of the pollutant. For CSIA it is essential to know the kinetic isotope effects (KIEs) pertinent to the dioxygenation mechanism of organic contaminants. Unfortunately, the range of KIEs of such reactions is poorly constrained although many dioxygenase enzymes with a broad substrate specificity have been reported. Dioxygenase enzymes usually exhibit complex reaction kinetics involving multiple substrates and substrate-specific binding modes which makes the determination of KIEs challenging. The goal of this study was to explore the magnitude and variability of 13C-, 2H-, and 15N-KIEs for the dioxygenation of one contaminant class, that is nitroaromatic contaminants (NACs). To this end, we investigated the C, H, and N isotope fractionation during the dioxygenation of nitrobenzene (NB), 2-nitrotoluene (2-NT), and 3-nitrotoluene (3-NT) by pure cultures, E. coli clones, cell extracts, and purified enzymes. From isotope fractionations measured in the substrates and reaction products, we determined dioxygenation KIEs for different combinations of the three substrates with nitrobenzene dioxygenase (NBDO) and 2-nitrotoluene dioxygenase (2NTDO). The 13C-, 2H-, and 15N-KIEs for the dioxygenation of NB by NBDO were consistent for all experimental systems considered (i.e., Comamonas sp. Strain JS765, E. coli clones, cell extracts of E. coli clones, and purified NBDO). This observation suggests that the isotope

  9. Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates. (United States)

    Thrivikraman, Greeshma; Madras, Giridhar; Basu, Bikramjit


    In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10(-9) to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10(-5) to 1 m) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and βIII tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown on electroconductive substrates.

  10. A broad distribution of the alternative oxidase in microsporidian parasites.

    Directory of Open Access Journals (Sweden)

    Bryony A P Williams


    Full Text Available Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX, a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1 as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

  11. Friction and Shear Strength at the Nanowire–Substrate Interfaces

    Directory of Open Access Journals (Sweden)

    Gu Yi


    Full Text Available Abstract The friction and shear strength of nanowire (NW–substrate interfaces critically influences the electrical/mechanical performance and life time of NW-based nanodevices. Yet, very few reports on this subject are available in the literature because of the experimental challenges involved and, more specifically no studies have been reported to investigate the configuration of individual NW tip in contact with a substrate. In this letter, using a new experimental method, we report the friction measurement between a NW tip and a substrate for the first time. The measurement was based on NW buckling in situ inside a scanning electron microscope. The coefficients of friction between silver NW and gold substrate and between ZnO NW and gold substrate were found to be 0.09–0.12 and 0.10–0.15, respectively. The adhesion between a NW and the substrate modified the true contact area, which affected the interfacial shear strength. Continuum mechanics calculation found that interfacial shear strengths between silver NW and gold substrate and between ZnO NW and gold substrate were 134–139 MPa and 78.9–95.3 MPa, respectively. This method can be applied to measure friction parameters of other NW–substrate systems. Our results on interfacial friction and shear strength could have implication on the AFM three-point bending tests used for nanomechanical characterisation.

  12. Cancer and the metastatic substrate (United States)

    Arvelo, Francisco; Sojo, Felipe; Cotte, Carlos


    Seventy percent of cancer patients have detectable metastases when they receive a diagnosis and 90% of cancer deaths result from metastases. These two facts emphasise the urgency for research to study the mechanisms and processes that enable metastasis. We need to develop a greater understanding of the cellular and molecular mechanisms that cause metastasis and also we need to do more. We must also consider the micro- and macro-environmental factors that influence this disease. Studying this environmental context has led us to update the ‘seed and soil’ hypothesis which dates back to the 19th century. This theory describes cancerous cells as seeds and the substrate as the soil in target organs though this may seem antiquated. Nonetheless, the tissue specificity that researchers have recently observed in metastatic colonisation supports the validity of the seed and soil theory. We now know that the metastatic potential of a tumour cell depends on multiple, reciprocal interactions between the primary tumour and distant sites. These interactions determine tumour progression. Studies of metastasis have allowed us to develop treatments that focus on therapeutic effectiveness. These new treatments account for the frequent metastasis of some tumours to target organs such as bones, lungs, brain, and liver. The purpose of this review is first to describe interactions between the cellular and molecular entities and the target organ tumour environment that enables metastasis. A second aim is to describe the complex mechanisms that mediate these interactions. PMID:28105072

  13. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples. (United States)

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne


    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

  14. Ultra-broad bandwidth parametric amplification at degeneracy. (United States)

    Limpert, J; Aguergaray, C; Montant, S; Manek-Hönninger, I; Petit, S; Descamps, D; Cormier, E; Salin, F


    We report on a novel approach of ultra-broad bandwidth parametric amplification around degeneracy. A bandwidth of up to 400 nm centered around 800 nm is amplified in a BBO crystal by using chirped pump pulses with a bandwitdth as broad as 10 nm. A supercontinuum signal is generated in a microstructured fiber, having to first order a quadratic chirp, which is necessary to ensure temporal overlap of the interacting waves over this broad bandwidth. Furthermore, we discuss the potential of this approach for an octave-spanning parametric amplification.

  15. A Novel Radiative Substrate Heater


    CEYLAN, Abdullah; Kaynak, Erdal; ÖZCAN, Şadan; Firat, Tezer


    In this work, we have developed a new low cost radiative substrate heater for deposition of high-Tc superconducting thin films. It has all the features required for the preparation of high quality superconducting thin films. It is possible to reach ~ 800 °C substrate temperature with it by using only one 250 W halogen projector bulb.

  16. Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate (United States)

    Linenberger, Kimberly J.; Bretz, Stacey Lowery


    Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

  17. Specific Phobias (United States)

    ... Mental Health This information in Spanish ( en español ) Specific phobias Treatment More information on specific phobias A specific ... targeted psychotherapy. Return to top More information on Specific phobias Explore other publications and websites Phobias (Copyright © American ...

  18. Soft substrates suppress droplet splashing

    CERN Document Server

    Howland, Christopher J; Style, Robert W; Castrejón-Pita, A A


    Droplets splash when they impact dry, flat substrates above a critical velocity that depends on parameters such as droplet size, viscosity and air pressure. We show that substrate stiffness also impacts the splashing threshold by imaging ethanol drops impacting silicone gels of different stiffnesses. Splashing is significantly suppressed: droplets on the softest substrates need over 70% more kinetic energy to splash than they do on rigid substrates. We show that splash suppression is likely to be due to energy losses caused by deformations of soft substrates during the first few microseconds of impact. We find that solids with Youngs modulus $\\lesssim O(10^5)$Pa suppress splashing, in agreement with simple scaling arguments. Thus materials like soft gels and elastomers can be used as simple coatings for effective splash prevention.

  19. AGN Broad Line Regions Scale with Bolometric Luminosity

    CERN Document Server

    Trippe, Sascha


    The masses of supermassive black holes in active galactic nuclei (AGN) can be derived spectroscopically via virial mass estimators based on selected broad optical/ultraviolet emission lines. These estimates commonly use the line width as a proxy for the gas speed and the monochromatic continuum luminosity as a proxy for the radius of the broad line region. However, if the size of the broad line region scales with bolometric rather than monochromatic AGN luminosity, mass estimates based on different emission lines will show a systematic discrepancy which is a function of the color of the AGN continuum. This has actually been observed in mass estimates based on H-alpha / H-beta and C IV lines, indicating that AGN broad line regions indeed scale with bolometric luminosity. Given that this effect seems to have been overlooked as yet, currently used single-epoch mass estimates are likely to be biased.

  20. Broad Spectrum Sanitizing Wipes with Food Additives Project (United States)

    National Aeronautics and Space Administration — Microcide proposes to develop novel multipurpose non-toxic sanitizing wipes that are aqueous based, have shelf life of 3-5 years, have broad spectrum microbicidal...

  1. Broad-Scale Genetic Diversity of Cannabis for Forensic Applications (United States)

    Dufresnes, Christophe; Jan, Catherine; Bienert, Friederike; Goudet, Jérôme; Fumagalli, Luca


    Cannabis (hemp and marijuana) is an iconic yet controversial crop. On the one hand, it represents a growing market for pharmaceutical and agricultural sectors. On the other hand, plants synthesizing the psychoactive THC produce the most widespread illicit drug in the world. Yet, the difficulty to reliably distinguish between Cannabis varieties based on morphological or biochemical criteria impedes the development of promising industrial programs and hinders the fight against narcotrafficking. Genetics offers an appropriate alternative to characterize drug vs. non-drug Cannabis. However, forensic applications require rapid and affordable genotyping of informative and reliable molecular markers for which a broad-scale reference database, representing both intra- and inter-variety variation, is available. Here we provide such a resource for Cannabis, by genotyping 13 microsatellite loci (STRs) in 1 324 samples selected specifically for fibre (24 hemp varieties) and drug (15 marijuana varieties) production. We showed that these loci are sufficient to capture most of the genome-wide diversity patterns recently revealed by NGS data. We recovered strong genetic structure between marijuana and hemp and demonstrated that anonymous samples can be confidently assigned to either plant types. Fibres appear genetically homogeneous whereas drugs show low (often clonal) diversity within varieties, but very high genetic differentiation between them, likely resulting from breeding practices. Based on an additional test dataset including samples from 41 local police seizures, we showed that the genetic signature of marijuana cultivars could be used to trace crime scene evidence. To date, our study provides the most comprehensive genetic resource for Cannabis forensics worldwide. PMID:28107530

  2. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy. (United States)

    Ma, Yuxiang; Chen, Haiyan; Su, Shanyuhan; Wang, Tong; Zhang, Congying; Fida, Guissi; Cui, Sisi; Zhao, Juan; Gu, Yueqing


    Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have th