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Sample records for amidases

  1. Kallikrein-like amidase activity in renal ischemia and reperfusion

    Directory of Open Access Journals (Sweden)

    M.D. Carattino

    2000-05-01

    Full Text Available We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 µg of Evans blue/g dry tissue in the basal condition and 1750 µg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage

  2. Amidase encapsulated O-carboxymethyl chitosan nanoparticles for vaccine delivery.

    Science.gov (United States)

    Smitha, K T; Sreelakshmi, M; Nisha, N; Jayakumar, R; Biswas, Raja

    2014-02-01

    This work reports the development of amidase encapsulated O-carboxymethyl chitosan nanoparticles (Ami-O-CMC NPs) of 300±50 nm size by ionic cross-linking method. The prepared Ami-O-CMC NPs had an encapsulation efficiency of 55.39%. Haemolysis assay and cytotoxicity studies proved the hemocompatibility and cytocompatibility of the prepared NPs. The sustained release of Ami from the NPs is expected to prolong its immunogenicity and in turn lead to development of better protective immunity against Staphylococcus aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Chemically modified carboxypeptidase Y with increased amidase activity

    International Nuclear Information System (INIS)

    Breddam, K.

    1984-01-01

    Treatment of carboxypeptidase Y with 14 C-iodoacetamide caused a drastic reduction in the peptidase activity towards FA-Phe-Leu-OH while the esterase activity towards FA-Phe-OMe, the amidase activity towards FA-Phe-NH 2 and the peptidyl amino acid amide hydrolase activity towards FA-Phe-Gly-NH 2 were much less affected. The loss of peptidase activity could be correlated with the incorporation of a single equivalent of reagent and it was demonstrated that the site of reaction was a methionyl residue, thus forming a sulfonium derivative. Analogous methionyl modifications were performed: carboxypeptidase Y modified with phenacylbromide hydrolysed substrates with bulky leaving groups in the P position, i.e. -OEt, -OBzl, -Gly-NH 2 ,-Gly-OH, and -Leu-OH, at reduced rates while substrates with small groups in that position, i.e. -OMe and -NH 2 , were hydrolysed with increased rates. These results indicate that the methionyl residue modified by phenacylbromide is located in the S binding site of the enzyme. Similar results were obtained with carboxypeptidase Y modified with m-nitrophen- acylbromide and p-nitrophenacylbromide. The increase in amidase activity and decrease in peptidyl amino acid amide hydrolase activity of carboxypeptidase Y following modification with phenacylbromide, m-nitrophenacylbromide, and p-nitrophenacylbromide was exploited in deamidation of peptide amides. These modified enzymes deamidated peptide amides with the exception of those containing a C-terminal glycyl or seryl residue in yields of 80-100% which is significantly higher than with unmodified carboxypeptidase Y. (author)

  4. Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in situ cascade system.

    Science.gov (United States)

    Cantarella, Laura; Gallifuoco, Alberto; Spera, Agata; Cantarella, Maria

    2013-08-01

    In this study the amidase kinetics of an in situ NHase/AMase cascade system was explored as a function of operational parameters such as temperature, substrate concentration and product formation. The results indicated that controlling amidase inactivation, during acrylonitrile bioconversion, makes it possible to recover the intermediate product of the two-step reaction in almost a pure form, without using purified enzyme. It has been demonstrated, in long-term experiments performed in continuous stirred UF-membrane bioreactors, that amidase is kinetically controlled by its proper substrate, depending on the structure, and by acrylonitrile. Using acrylamide, AMase-stability is temperature dependent (5°C, kd=0.008 h(-1); 30°C kd=0.023 h(-1)). Using benzamide, amidase is thermally stable up to 50°C and no substrate inhibition/inactivation occurs. With acrylonitrile, AMase-activity and -stability remain unchanged at concentrations <200 mM but at 200 mM, 35°C, after 70 h process, 90% irreversible inactivation occurs as no AMase-activity on benzamide revives. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Biotransformation of benzonitrile herbicides via the nitrile hydratase-amidase pathway in rhodococci

    Czech Academy of Sciences Publication Activity Database

    Veselá, Alicja Barbara; Pelantová, Helena; Šulc, Miroslav; Macková, M.; Lovecká, P.; Thimová, P.; Pasquarelli, F.; Pičmanová, Martina; Pátek, Miroslav; Bhalla, T. C.; Martínková, Ludmila

    2012-01-01

    Roč. 39, č. 12 (2012), s. 1811-1819 ISSN 1367-5435 R&D Projects: GA MŠk OC09046; GA ČR(CZ) GAP504/11/0394; GA ČR GD305/09/H008 Keywords : Nitrile hydratase * Amidase * Benzonitrile herbicides Subject RIV: EE - Microbiology, Virology Impact factor: 2.321, year: 2012

  6. Biotransformation of nitriles to hydroxamic acids via a nitrile hydratase–amidase cascade reaction

    Czech Academy of Sciences Publication Activity Database

    Vejvoda, Vojtěch; Martínková, Ludmila; Veselá, Alicja Barbara; Kaplan, Ondřej; Lutz-Wahl, S.; Fischer, L.; Uhnáková, Bronislava

    2011-01-01

    Roč. 71, 1-2 (2011), s. 51-55 ISSN 1381-1177 R&D Projects: GA MŠk(CZ) LC06010; GA MŠk OC09046 Institutional research plan: CEZ:AV0Z50200510 Keywords : Nitrile hydratase * Rhodococcus erythropolis * Amidase Subject RIV: EE - Microbiology, Virology Impact factor: 2.735, year: 2011

  7. Amidase-catalyzed production of nicotinic acid in batch and continuous stirred membrane reactors

    Czech Academy of Sciences Publication Activity Database

    Cantarella, M.; Cantarella, L.; Gallifuoco, A.; Intellini, R.; Kaplan, Ondřej; Spera, A.; Martínková, Ludmila

    2008-01-01

    Roč. 42, č. 3 (2008), s. 222-229 ISSN 0141-0229 R&D Projects: GA MŠk OC D25.002 Institutional research plan: CEZ:AV0Z50200510 Keywords : amidase * nicotinic acid bioproduction * temperature dependence Subject RIV: EE - Microbiology, Virology Impact factor: 2.375, year: 2008

  8. Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

    Science.gov (United States)

    Rucká, Lenka; Volkova, Olga; Pavlík, Adam; Kaplan, Ondřej; Kracík, Martin; Nešvera, Jan; Martínková, Ludmila; Pátek, Miroslav

    2014-06-01

    Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.

  9. Immobilization of fungal nitrilase and bacterial amidase – two enzymes working in accord

    Czech Academy of Sciences Publication Activity Database

    Vejvoda, Vojtěch; Kaplan, Ondřej; Kubáč, David; Křen, Vladimír; Martínková, Ludmila

    2006-01-01

    Roč. 24, č. 6 (2006), s. 414-418 ISSN 1024-2422 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GA203/05/2267; GA MŠk 1P05OC073; GA MŠk OC D25.001 Institutional research plan: CEZ:AV0Z50200510 Keywords : amidase * 4-cyanopyridine * isonicotinic acid Subject RIV: EE - Microbiology, Virology Impact factor: 1.437, year: 2006

  10. Increasing plant growth by modulating omega-amidase expression in plants

    Science.gov (United States)

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2015-06-30

    The present disclosure relates to compositions and methods for increasing the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules in plants by modulating levels of .omega.-amidase to increase nitrogen use efficiency, resulting in enhanced growth, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.

  11. Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase.

    Science.gov (United States)

    Lin, Zhi-Jian; Zheng, Ren-Chao; Lei, Li-Hua; Zheng, Yu-Guo; Shen, Yin-Chu

    2011-06-01

    Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

    Science.gov (United States)

    Buchholz, Klaus

    2016-05-01

    Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that

  13. Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

    Science.gov (United States)

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-04-18

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

  14. Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family

    International Nuclear Information System (INIS)

    Lee, Saeyoung; Park, Eun-Hye; Ko, Hyeok-Jin; Bang, Won Gi; Kim, Hye-Yeon; Kim, Kyoung Heon; Choi, In-Geol

    2015-01-01

    The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/β fold class, exhibiting an open twisted β-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad, is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr"1"3"6 and Thr"3"3"0) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys"8"4 as the catalytic base to polarize the Ser"1"8"7 nucleophile in the catalytic triad. - Highlights: • We determined the first structure of a bacterial aryl acylamidase (EC 3.5.1.13). • Structure revealed spatially distinct architecture of the substrate-binding pocket. • Hydrogen-bonding with Tyr"1"3"6 and Thr"3"3"0 mediates ligand-binding and substrate.

  15. Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage*

    Science.gov (United States)

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-01-01

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen. PMID:24599952

  16. Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Saeyoung; Park, Eun-Hye; Ko, Hyeok-Jin; Bang, Won Gi [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seoungbuk-Gu, Seoul, 136-713 (Korea, Republic of); Kim, Hye-Yeon [Protein Structure Research Team, Korea Basic Science Institute, Ochang, Chungbuk, 363-883 (Korea, Republic of); Kim, Kyoung Heon [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seoungbuk-Gu, Seoul, 136-713 (Korea, Republic of); Choi, In-Geol, E-mail: igchoi@korea.ac.kr [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seoungbuk-Gu, Seoul, 136-713 (Korea, Republic of)

    2015-11-13

    The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/β fold class, exhibiting an open twisted β-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad, is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr{sup 136} and Thr{sup 330}) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys{sup 84} as the catalytic base to polarize the Ser{sup 187} nucleophile in the catalytic triad. - Highlights: • We determined the first structure of a bacterial aryl acylamidase (EC 3.5.1.13). • Structure revealed spatially distinct architecture of the substrate-binding pocket. • Hydrogen-bonding with Tyr{sup 136} and Thr{sup 330} mediates ligand-binding and substrate.

  17. High-yield continuous production of nicotinic acid via nitrile hydratase–amidase cascade reactions using cascade CSMRs

    Czech Academy of Sciences Publication Activity Database

    Cantarella, L.; Gallifuoco, A.; Malandra, A.; Martínková, Ludmila; Spera, A.; Cantarella, M.

    2011-01-01

    Roč. 48, 4-5 (2011), 345-350 ISSN 0141-0229 R&D Projects: GA MŠk OC09046 Institutional research plan: CEZ:AV0Z50200510 Keywords : Nitrile hydratase-amidase cascade system * 3-Cyanopyridine bioconversion * Nicotinic acid Subject RIV: EE - Microbiology, Virology Impact factor: 2.367, year: 2011

  18. Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes

    Czech Academy of Sciences Publication Activity Database

    Rucká, Lenka; Volkova, Olga; Pavlík, Adam; Kaplan, Ondřej; Kracík, M.; Nešvera, Jan; Martínková, Ludmila; Pátek, Miroslav

    2014-01-01

    Roč. 105, č. 6 (2014), s. 1179-1190 ISSN 0003-6072 R&D Projects: GA MŠk(CZ) LC06010; GA ČR(CZ) GAP504/11/0394 Institutional support: RVO:61388971 Keywords : Rhodococcus erythropolis * Amidase * Nitrile hydratase Subject RIV: EE - Microbiology, Virology Impact factor: 1.806, year: 2014

  19. The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing

    International Nuclear Information System (INIS)

    Agarkar, Vinod B.; Kimani, Serah W.; Cowan, Donald A.; Sayed, Muhammed F.-R.; Sewell, B. Trevor

    2006-01-01

    The amidase from G. pallidus RAPc8, a moderate thermophile, converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned, expressed and purified, and then crystallized using the hanging-drop vapour-diffusion method. The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 Å resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4 2 32, with unit-cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved

  20. Structure-guided functional characterization of DUF1460 reveals a highly specific NlpC/P60 amidase family.

    Science.gov (United States)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine; 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

    2014-12-02

    GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predicted in silico based on structural and bioinformatics data, and subsequently were characterized experimentally. Further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines another amidase family. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase inhibition.

    Directory of Open Access Journals (Sweden)

    Jay M West

    Full Text Available The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA, with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. 1-Isothiocyanatopentadecane (AM9023 was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-ylmethyl-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701 and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone, inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126 of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.

  2. AmiD Is a Novel Peptidoglycan Amidase in Wolbachia Endosymbionts of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Miriam Wilmes

    2017-08-01

    Full Text Available Wolbachia endobacteria are obligate intracellular bacteria with a highly reduced genome infecting many arthropod and filarial species, in which they manipulate arthropod reproduction to increase their transmission and are essential for nematode development and survival. The Wolbachia genome encodes all enzymes required for the synthesis of the cell wall building block lipid II, although a peptidoglycan-like structure has not been detected. Despite the ability to synthesize lipid II, Wolbachia from arthropods and nematodes have only a subset of genes encoding enzymes involved in the periplasmic processing of lipid II and peptidoglycan recycling, with arthropods having two more than nematodes. We functionally analyzed the activity of the putative cell wall hydrolase AmiD from the Wolbachia endosymbiont of Drosophila melanogaster, an enzyme not encoded by the nematode endobacteria. Wolbachia AmiD has Zn2+-dependent amidase activity and cleaves intact peptidoglycan, monomeric lipid II and anhydromuropeptides, substrates that are generated during bacterial growth. AmiD may have been maintained in arthropod Wolbachia to avoid host immune recognition by degrading cell wall fragments in the periplasm. This is the first description of a wolbachial lipid II processing enzyme putatively expressed in the periplasm.

  3. Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

    Directory of Open Access Journals (Sweden)

    Charlotte Kürten

    2016-06-01

    Full Text Available The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

  4. Effect of mass transfer in a recirculation batch reactor system for immobilized penicillin amidase.

    Science.gov (United States)

    Park, J M; Choi, C Y; Seong, B L; Han, M H

    1982-10-01

    The effect of external mass transfer resistance on the overall reaction rate of the immobilized whole cell penicillin amidase of E. coli in a recirculation batch reactor was investigated. The internal diffusional resistance was found negligible as indicated by the value of effectiveness factor, 0.95. The local environmental change in a column due to the pH drop was successfully overcome by employing buffer solution. The reaction rate was measured by pH-stat method and was found to follow the simple Michaelis-Menten law at the initial stage of the reaction. The values of the net reaction rate experimentally determined were used to calculate the substrate concentration at the external surface of the catalyst pellet and then to calculate the mass transfer coefficient, k(L), at various flow rates and substrate concentrations. The correlation proposed by Chilton and Colburn represented adequately the experimental data. The linear change of log j(D) at low log N(Re) with negative slope was ascribed to the fact that the external mass transfer approached the state of pure diffusion in the limit of zero superficial velocity.

  5. Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp. PCC 7120

    Directory of Open Access Journals (Sweden)

    Jan Bornikoel

    2017-09-01

    Full Text Available Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N2-fixing heterocysts and CO2-fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N-acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell–cell communication in Nostoc punctiforme. This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2, were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme, because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell–cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD.

  6. Enzyme-substrate binding landscapes in the process of nitrile biodegradation mediated by nitrile hydratase and amidase.

    Science.gov (United States)

    Zhang, Yu; Zeng, Zhuotong; Zeng, Guangming; Liu, Xuanming; Chen, Ming; Liu, Lifeng; Liu, Zhifeng; Xie, Gengxin

    2013-08-01

    The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme-substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and L-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme's binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.

  7. Nicotinic acid bio-production by Microbacterium imperiale CBS 489-74. Effect of 3-cyanopyridine and temperature on amidase activity

    Czech Academy of Sciences Publication Activity Database

    Cantarella, M.; Cantarella, L.; Gallifuoco, A.; Spera, A.; Martínková, Ludmila

    2012-01-01

    Roč. 47, č. 7 (2012), s. 1192-1196 ISSN 1359-5113 R&D Projects: GA MŠk OC09046 Keywords : Nitrile hydratase-amidase cascade system * 3-Cyanopyridine inactivation * 3-Cyanopyridine inhibition Subject RIV: CE - Biochemistry Impact factor: 2.414, year: 2012

  8. Application of continuous stirred membrane reactor to 3-cyanopyridine bioconversion using the nitrile hydratase–amidase cascade system of Microbacterium imperiale CBS 498-74

    Czech Academy of Sciences Publication Activity Database

    Cantarella, L.; Gallifuoco, A.; Malandra, A.; Martínková, Ludmila; Pasquarelli, F.; Spera, A.; Cantarella, M.

    2010-01-01

    Roč. 47, č. 3 (2010), s. 64-70 ISSN 0141-0229 R&D Projects: GA MŠk OC09046 Institutional research plan: CEZ:AV0Z50200510 Keywords : Nitrile hydratase-amidase cascade system * 3-Cyanopyridine bioconversion * Nicotinamide Subject RIV: EE - Microbiology, Virology Impact factor: 2.287, year: 2010

  9. In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB

    DEFF Research Database (Denmark)

    Hediger, Martin Robert; De Vico, Luca; Rannes, Julie Bille

    2013-01-01

    Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., 2012) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barriers......, the qualitative activity of 15 out of 22 mutants is correctly predicted. The method identifies four of the six most active mutants with ≥3-fold wild type activity and seven out of the eight least active mutants with ≤0.5-fold wild type activity. The method is further used to screen all sterically possible (386......) double-, triple- and quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified....

  10. Enabling cell-cell communication via nanopore formation: structure, function and localization of the unique cell wall amidase AmiC2 of Nostoc punctiforme.

    Science.gov (United States)

    Büttner, Felix M; Faulhaber, Katharina; Forchhammer, Karl; Maldener, Iris; Stehle, Thilo

    2016-04-01

    To orchestrate a complex life style in changing environments, the filamentous cyanobacterium Nostoc punctiforme facilitates communication between neighboring cells through septal junction complexes. This is achieved by nanopores that perforate the peptidoglycan (PGN) layer and traverse the cell septa. The N-acetylmuramoyl-l-alanine amidase AmiC2 (Npun_F1846; EC 3.5.1.28) in N. punctiforme generates arrays of such nanopores in the septal PGN, in contrast to homologous amidases that mediate daughter cell separation after cell division in unicellular bacteria. Nanopore formation is therefore a novel property of AmiC homologs. Immunofluorescence shows that native AmiC2 localizes to the maturing septum. The high-resolution crystal structure (1.12 Å) of its catalytic domain (AmiC2-cat) differs significantly from known structures of cell splitting and PGN recycling amidases. A wide and shallow binding cavity allows easy access of the substrate to the active site, which harbors an essential zinc ion. AmiC2-cat exhibits strong hydrolytic activity in vitro. A single point mutation of a conserved glutamate near the zinc ion results in total loss of activity, whereas zinc removal leads to instability of AmiC2-cat. An inhibitory α-helix, as found in the Escherichia coli AmiC(E. coli) structure, is absent. Taken together, our data provide insight into the cell-biological, biochemical and structural properties of an unusual cell wall lytic enzyme that generates nanopores for cell-cell communication in multicellular cyanobacteria. The novel structural features of the catalytic domain and the unique biological function of AmiC2 hint at mechanisms of action and regulation that are distinct from other amidases. The AmiC2-cat structure has been deposited in the Protein Data Bank under accession number 5EMI. © 2016 Federation of European Biochemical Societies.

  11. Transition state analogue imprinted polymers as artificial amidases for amino acid p-nitroanilides: morphological effects of polymer network on catalytic efficiency.

    Science.gov (United States)

    Mathew, Divya; Thomas, Benny; Devaky, K S

    2017-11-13

    The morphology of the polymer network - porous/less porous - plays predominant role in the amidase activities of the polymer catalysts in the hydrolytic reactions of amino acid p-nitroanilides. Polymers with the imprints of stable phosphonate analogue of the intermediate of hydrolytic reactions were synthesized as enzyme mimics. Molecular imprinting was carried out in thermodynamically stable porogen dimethyl sulphoxide and unstable porogen chloroform, to investigate the morphological effects of polymers on catalytic amidolysis. It was found that the medium of polymerization has vital influence in the amidase activities of the enzyme mimics. The morphological studies of the polymer catalysts were carried out by scanning electron microscopy and Bruner-Emmett-Teller analysis. The morphology of the polymer catalysts and their amidase activities are found to be dependent on the composition of reaction medium. The polymer catalyst prepared in dimethyl sulphoxide is observed to be efficient in 1:9 acetonitrile (ACN)-Tris HCl buffer and that prepared in chloroform is noticed to be stereo specifically and shape-selectively effective in 9:1 ACN-Tris HCl buffer. The solvent memory effect in catalytic amidolysis was investigated using the polymer prepared in acetonitrile.

  12. Amidase-responsive controlled release of antitumoral drug into intracellular media using gluconamide-capped mesoporous silica nanoparticles

    Science.gov (United States)

    Candel, Inmaculada; Aznar, Elena; Mondragón, Laura; Torre, Cristina De La; Martínez-Máñez, Ramón; Sancenón, Félix; Marcos, M. Dolores; Amorós, Pedro; Guillem, Carmen; Pérez-Payá, Enrique; Costero, Ana; Gil, Salvador; Parra, Margarita

    2012-10-01

    MCM-41 silica nanoparticles were used as inorganic scaffolding to prepare a nanoscopic-capped hybrid material S1, which was able to release an entrapped cargo in the presence of certain enzymes, whereas in the absence of enzymes, a zero release system was obtained. S1 was prepared by loading nanoparticles with Safranine O dye and was then capped with a gluconamide derivative. In the absence of enzymes, the release of the dye from the aqueous suspensions of S1 was inhibited as a result of the steric hindrance imposed by the bulky gluconamide derivative, the polymerized gluconamide layer and the formation of a dense hydrogen-bonded network around the pore outlets. Upon the addition of amidase and pronase enzymes, delivery of Safranine O dye was observed due to the enzymatic hydrolysis of the amide bond in the anchored gluconamide derivative. S1 nanoparticles were not toxic for cells, as demonstrated by cell viability assays using HeLa and MCF-7 cell lines, and were associated with lysosomes, as shown by confocal microscopy. Finally, the S1-CPT material loaded with the cytotoxic drug camptothecin and capped with the gluconamide derivative was prepared. The HeLa cells treated with S1-CPT underwent cell death as a result of material internalization, and of the subsequent cellular enzyme-mediated hydrolysis and aperture of the molecular gate, which induced the release of the camptothecin cargo.

  13. Latin-American Co-operation in Biotechnology Programme: Industrial penicillin amidase for 6 amino penicillanic acid production

    Directory of Open Access Journals (Sweden)

    Dolly Montoya C.

    2001-07-01

    Full Text Available This work evaluates technological and economic transference related to the Production of Penicillin Amidase for use in 6 Amine Penicillanic acid (6-APA Production Project, wich is a part of the United Nations1 Regional Biotechnology Programme for Latin America and the Caribbean. This paper analyses the evolution of international cooperation by looking at the project's development. The methodology used includes analysis of: the project's development; participant and budgetary goals; results; copyright; project conditions; and sale of biocatalyst and 6-APA. All technical objectives were achieved; international co-operation, as well as co-operation between Industry and University were successful. Technological transference to the pilot plant was effective; many students involved in the project were simultaneously taking M.Sc. and Ph.D's courses. Nevertheless, neither the technology necessary for the biocatalyst's manufacture nor the biocatalyst itself were used. Analysis of the project has provided some orientation concerning those internal and external problems which arose during the development and sale of biotechnology in our countries and has tried to propose some alternatives for taking advantage of international co-operation.

  14. Cell wall amidase AmiC1 is required for cellular communication and heterocyst development in the cyanobacterium Anabaena PCC 7120 but not for filament integrity.

    Science.gov (United States)

    Berendt, Susanne; Lehner, Josef; Zhang, Yao Vincent; Rasse, Tobias M; Forchhammer, Karl; Maldener, Iris

    2012-10-01

    Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.

  15. Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Waard, P. de; Koorevaar, A.; Kamerling, J.P.

    1991-01-01

    The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F (PNGase- F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and

  16. Facilitated stimulus-response associative learning and long-term memory in mice lacking the NTAN1 amidase of the N-end rule pathway.

    Science.gov (United States)

    Balogh, S A; McDowell, C S; Tae Kwon, Y; Denenberg, V H

    2001-02-23

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Inactivation of the NTAN1 gene encoding the asparagine-specific N-terminal amidase in mice results in impaired spatial memory [26]. The studies described here were designed to further characterize the effects upon learning and memory of inactivating the NTAN1 gene. NTAN1-deficient mice were found to be better than wild-type mice on black-white and horizontal-vertical discrimination learning. They were also better at 8-week Morris maze retention testing when a reversal trial was not included in the testing procedures. In all three tasks NTAN1-deficient mice appeared to use a strong win-stay strategy. It is concluded that inactivating the asparagine-specific branch of the N-end rule pathway in mice results in impaired spatial learning with concomitant compensatory restructuring of the nervous system in favor of non-spatial (stimulus-response) learning.

  17. Lytr, a phage-derived amidase is most effective in induced lysis of Lactococcus lactis compared with other lactococcal amidases and glucosaminidases

    NARCIS (Netherlands)

    Steen, Anton; van Schalkwijk, Saskia; Buist, Girbe; Twigt, Marja; Szeliga, Monika; Meijer, Wilco; Kuipers, Oscar P.; Kok, Jan; Hugenholtz, Jeroen

    In the genome of Lactococcus lactis IL1403 five genes encoding peptidoglycan hydrolases are present: four glucosaminidases (acmA, acmB, acmC and acmD) and an endopeptidase (yjgB). Genes for six prophage lysins have also been identified. The genes acmB, acmC, acmD, yjgB and the lysin lytR of prophage

  18. Biotransformation of benzonitrile herbicides via the nitrile hydratase-amidase pathway in rhodococci.

    Science.gov (United States)

    Veselá, Alicja B; Pelantová, Helena; Sulc, Miroslav; Macková, Martina; Lovecká, Petra; Thimová, Markéta; Pasquarelli, Fabrizia; Pičmanová, Martina; Pátek, Miroslav; Bhalla, Tek Chand; Martínková, Ludmila

    2012-12-01

    The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.

  19. Studies on peptide amidase-catalysed C-terminal peptide amidation in organic media with respect to its substrate specificity

    Czech Academy of Sciences Publication Activity Database

    Čeřovský, Václav; Kula, M. R.

    2001-01-01

    Roč. 33, - (2001), s. 183-187 ISSN 0885-4513 R&D Projects: GA ČR GA203/99/1458 Keywords : enzymic amidation * peptide amides * peptide synthesis Subject RIV: CC - Organic Chemistry Impact factor: 1.408, year: 2001

  20. Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

    Science.gov (United States)

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to iden...

  1. Recombinant Expression of a Genome-encoded N-acetylmuramoyl-L-alanine Amidase that Synergistically Lyses Listeria monocytogenes Biofilms with a Protease

    Science.gov (United States)

    Listeria monocytogenes plays a significant role in human food-borne disease caused by eating food contaminated with the bacterium and although incidence is low it is a leading cause of life-threatening, bacterial food-borne disease in humans. L. monocytogenes serotypes 1/2a and 4b can form mixed-cu...

  2. ORF Alignment: NC_004663 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ne ... amidase [Bacteroides thetaiotaomicron VPI-5482] ... Length = 145 ... Query: 3 ... KQRNISLIVVHCTASRCTSDLT...PPSLDAMHKRQGFTECGYHYYITKDGRIHHMRDITKIG 62 ... KQRNISLIVVHCTASRCTSDLT...PPSLDAMHKRQGFTECGYHYYITKDGRIHHMRDITKIG Sbjct: 1 ... KQRNISLIVVHCTASRCTSDLTPPSLDAMHKRQGFTECGYHYYITKDGRIHH

  3. Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide.

    Science.gov (United States)

    Gregoriou, M; Brown, P R; Tata, R

    1977-11-01

    Pseudomonas aeruginosa AI 3 was able to grow in medium containing acetanilide (N-phenylacetamide) as a carbon source when NH4+ was the nitrogen source but not when urea was the nitrogen source. AIU mutants isolated from strain AI 3 grew on either medium. Urease levels in bacteria grown in the presence of urea were 10-fold lower when NH4+ or acetanilide was also in the medium, but there were no apparent differences in urease or its synthesis between strain AI 3 and mutant AIU 1N. The first metabolic step in the acetanilide utlization is catalyzed by an amidase. Amidases in several AIU strains showed altered physiochemical properties. Urea inhibited amidase in a time-dependent reaction, but the rates of the inhibitory reaction with amidases from the AIU mutants were slower than with AI 3 amidase. The purified amidase from AIU 1N showed a marked difference in its pH/activity profile from that obtained with purified AI 3 amidase. These observations indicate that the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene; this was confirmed for strain AIU 1N by transduction.

  4. Characterisation of the nitrile biocatalytic activity of rhodococcus rhodochrous ATCC BAA-870

    CSIR Research Space (South Africa)

    Frederick, J

    2006-10-01

    Full Text Available rhodochrous ATCC BAA-870, was explored. The biocatalyst expressed a two enzyme system with sequential nitrile-converting activity: nitrile hydratase and amidase. This biocatalytic nitrile hydrolysis affords valuable applications in industry, including...

  5. A Novel Chimeric Endolysin with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Haddad Kashani, Hamed; Fahimi, Hossein; Dasteh Goli, Yasaman; Moniri, Rezvan

    2017-01-01

    Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Subsequent affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of ≥1 μg/mL of the purified protein have significant antibacterial activity against S. aureus MRSA 252 cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA 252 cell counts at the concentration of 10 μg/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC) index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis , and enterococcus . However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis . Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.

  6. Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7- aminodeacetoxycephalosporanic acid production

    DEFF Research Database (Denmark)

    Robin, Jarno Jacky Christian; Jakobsen, M.; Beyer, M.

    2001-01-01

    The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this pr......The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase...

  7. A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: a study of both kinetics of inactivation and specifics of interaction with anionic polymers

    Science.gov (United States)

    A staphylolytic fusion protein (K-L) was created, harboring three unique lytic activities comprised of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of K-L...

  8. Microbial nitrilases: versatile, spiral forming, industrial enzymes

    CSIR Research Space (South Africa)

    Thuku, RN

    2009-03-01

    Full Text Available such case is the NAD+ synthetase from Mycobacterium tuberculosis (Bellinzoni et al., 2005). This enzyme relies on an associated amino-terminal amidase domain in order to utilize glutamine as a source of nitrogen and liberate ammonia which is required...

  9. Acid Ceramidase in Melanoma

    DEFF Research Database (Denmark)

    Realini, Natalia; Palese, Francesca; Pizzirani, Daniela

    2016-01-01

    Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphing...

  10. Biotransformation of 3-substituted methyl (R,S)-4-cyanobutanoates with nitrile- and amide-converting biocatalysts

    Czech Academy of Sciences Publication Activity Database

    Martínková, Ludmila; Klempier, N.; Bardakji, J.; Kandelbauer, A.; Ovesná, M.; Podařilová, T.; Kuzma, Marek; Přepechalová, Irena; Griengl, G.; Křen, Vladimír

    2001-01-01

    Roč. 14, - (2001), s. 95-99 ISSN 1381-1177 R&D Projects: GA AV ČR IAA4020802 Institutional research plan: CEZ:AV0Z5020903 Keywords : Rhodococcus equi * nitrile hydratase * amidase Subject RIV: EE - Microbiology, Virology Impact factor: 1.408, year: 2001

  11. GenBank blastx search result: AK059236 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059236 001-024-F01 AB016078.1 Rhodococcus sp. N-771 genes for nitrile hydratase r...egulator 2 and 1, amidase, nitrile hydratase alpha and beta subunits and nitrile hydratase activator, complete cds.|BCT BCT 8e-18 +3 ...

  12. GenBank blastx search result: AK243402 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243402 J100064O18 AB016078.1 AB016078 Rhodococcus sp. N-771 genes for nitrile hyd...ratase regulator 2 and 1, amidase, nitrile hydratase alpha and beta subunits and nitrile hydratase activator, complete cds. BCT 1e-27 1 ...

  13. Identification of novel open reading frames from metagenomic libraries generated from extremophilic organisms: application of metagenomics and high throughput screening for novel enzyme isolation

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2006-10-01

    Full Text Available : Olive Oil / Rhodamine B Tributyrin Fluorescence under UV Secondary Screening Colorimet- ric - PNP-ester substrates (C2, C4, C18); Substrate, Temperature and pH Profiling 16S ribosomal RNA gene (DNA level) Amidase ATP-dependant protease Beta...

  14. Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Hård, K.; Mekking, A.; Damm, J.B.L.; Kamerling, J.P.; Boer, W. de; Wijnands, R.A.

    1990-01-01

    Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides were separated from

  15. Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation

    Czech Academy of Sciences Publication Activity Database

    Dzyuba, V.; Słowińska, M.; Cosson, J.; Ciereszko, A.; Boryshpolets, S.; Štěrba, Ján; Rodina, M.; Linhart, O.; Dzyuba, B.

    2016-01-01

    Roč. 42, č. 6 (2016), s. 1755-1766 ISSN 0920-1742 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : amidase activity * anti-proteolytic activity * casein and gelatin zymography * siberian sturgeon sperm * spermatozoon maturation * sterlet sperm Subject RIV: ED - Physiology Impact factor: 1.647, year: 2016

  16. Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

    Science.gov (United States)

    Migliore, Marco; Pontis, Silvia; Fuentes de Arriba, Angel Luis; Realini, Natalia; Torrente, Esther; Armirotti, Andrea; Romeo, Elisa; Di Martino, Simona; Russo, Debora; Pizzirani, Daniela; Summa, Maria; Lanfranco, Massimiliano; Ottonello, Giuliana; Busquet, Perrine; Jung, Kwang-Mook; Garcia-Guzman, Miguel; Heim, Roger; Scarpelli, Rita; Piomelli, Daniele

    2016-09-05

    Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Biotransformation of heterocyclic dinitriles by Rhodococcus erythropolis and fungal nitrilases

    Czech Academy of Sciences Publication Activity Database

    Vejvoda, Vojtěch; Šveda, Ondřej; Kaplan, Ondřej; Přikrylová, Věra; Elišáková, Veronika; Himl, Michal; Kubáč, David; Pelantová, Helena; Kuzma, Marek; Křen, Vladimír; Martínková, Ludmila

    2007-01-01

    Roč. 29, č. 7 (2007), s. 1119-1124 ISSN 0141-5492 R&D Projects: GA AV ČR IAA500200708; GA MŠk LC06010; GA MŠk OC D25.001 Institutional research plan: CEZ:AV0Z50200510 Keywords : amidase * arpegillus nniger * heterocyclic Subject RIV: EE - Microbiology, Virology Impact factor: 1.222, year: 2007

  18. Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats

    Czech Academy of Sciences Publication Activity Database

    Kubáč, David; Čejková, A.; Masák, J.; Jirků, V.; Lemaire, M.; Gallienne, E.; Bolte, J.; Stloukal, R.; Martínková, Ludmila

    2006-01-01

    Roč. 39, - (2006), s. 59-61 ISSN 1381-1177 R&D Projects: GA ČR GA203/05/2267; GA MŠk OC D25.001; GA AV ČR IAA4020213 Institutional research plan: CEZ:AV0Z50200510 Keywords : nitrile hydratase * amidase * nitriles Subject RIV: EE - Microbiology, Virology Impact factor: 2.149, year: 2006

  19. The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites

    Directory of Open Access Journals (Sweden)

    Vivek A. Hariharan

    2017-03-01

    Full Text Available Many enzymes make “mistakes”. Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH and mitochondrial malate dehydrogenase (mMDH slowly catalyze the reduction of 2-oxoglutarate (2-OG to the oncometabolite l-2-hydroxyglutarate (l-2-HG. l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine. We show here that human glutamine synthetase (GS catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM. We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain “mistakes” of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

  20. The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites.

    Science.gov (United States)

    Hariharan, Vivek A; Denton, Travis T; Paraszcszak, Sarah; McEvoy, Kyle; Jeitner, Thomas M; Krasnikov, Boris F; Cooper, Arthur J L

    2017-03-30

    Many enzymes make "mistakes". Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain "mistakes" of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

  1. Pharmacological characterization of hydrolysis-resistant analogs of oleoylethanolamide with potent anorexiant properties

    OpenAIRE

    Astarita, G; Di Giacomo, B; Gaetani, S; Oveisi, F; Compton, TR; Rivara, S; Tarzia, G; Mor, M; Piomelli, D

    2006-01-01

    Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-α (PPAR-α). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-α with high potency in...

  2. Examination pf Potential Anti-Tumor Activity of N-Thiolated B-Lactam Antibiotics in Nude Mice Bearing Human Breast Tumors

    Science.gov (United States)

    2005-08-01

    their mechanism of action, production of reactive oxygen species (ROS), which leads to toxicity of the cardiomyocytes and subsequent chronic and acute...gemcitabine, and the purine analogs, such as 6- mercaptopurine and 6-thioguanine. These analogs substitute for nucleic acid bases in both DNA and RNA...Bioorg Med Chem Lett 12, 2229-2231 (2002) 14. Holt, R.J. and G.T. Stewart: Production of Amidase and beta-Lactamase by Bacteria. J Gen Microbiol 36

  3. Selection and screening for enzymes of nitrile metabolism

    Czech Academy of Sciences Publication Activity Database

    Martínková, Ludmila; Vejvoda, Vojtěch; Křen, Vladimír

    2008-01-01

    Roč. 133, č. 3 (2008), s. 318-326 ISSN 0168-1656 R&D Projects: GA AV ČR IAA500200708; GA ČR GA203/05/2267; GA MŠk(CZ) LC06010; GA MŠk OC 171 Institutional research plan: CEZ:AV0Z50200510 Keywords : nitrilase * nitrile hydratase * amidase Subject RIV: CE - Biochemistry Impact factor: 2.748, year: 2008

  4. Enzymatic assays for the diagnosis of bradykinin-dependent angioedema.

    Directory of Open Access Journals (Sweden)

    Federica Defendi

    Full Text Available BACKGROUND: The kinins (primarily bradykinin, BK represent the mediators responsible for local increase of vascular permeability in hereditary angioedema (HAE, HAE I-II associated with alterations of the SERPING1 gene and HAE with normal C1-Inhibitor function (HAE-nC1INH. Besides C1-Inhibitor function and concentration, no biological assay of kinin metabolism is actually available to help physicians for the diagnosis of angioedema (AE. We describe enzymatic tests on the plasma for diagnosis of BK-dependent AE. METHODS: The plasma amidase assays are performed using the Pro-Phe-Arg-p-nitroanilide peptide substrate to evaluate the spontaneous amidase activity and the proenzyme activation. We analyzed data of 872 patients presenting with BK-dependent AE or BK-unrelated diseases, compared to 303 controls. Anti-high MW kininogen (HK immunoblot was achieved to confirm HK cleavage in exemplary samples. Reproducibility, repeatability, limit of blank, limit of detection, precision, linearity and receiver operating characteristics (ROC were used to calculate the diagnostic performance of the assays. RESULTS: Spontaneous amidase activity was significantly increased in all BK-dependent AE, associated with the acute phase of disease in HAE-nC1INH, but preserved in BK-unrelated disorders. The increase of the amidase activity was associated to HK proteolysis, indicating its relevance to identify kininogenase activity. The oestrogens, known for precipitating AE episodes, were found as triggers of enzymatic activity. Calculations from ROC curves gave the optimum diagnostic cut-off for women (9.3 nmol⋅min(-1⋅mL(-1, area under curve [AUC] 92.1%, sensitivity 80.0%, and specificity 90.1% and for men (6.6 nmol·min(-1⋅mL(-1, AUC 91.0%, sensitivity 87.0% and specificity 81.2%. CONCLUSION: The amidase assay represents a diagnostic tool to help physicians in the decision to distinguish between BK-related and -unrelated AE.

  5. Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

    Czech Academy of Sciences Publication Activity Database

    Kubáč, David; Kaplan, Ondřej; Elišáková, Veronika; Pátek, Miroslav; Vejvoda, Vojtěch; Slámová, Kristýna; Tóthová, A.; Lemaire, M.; Gallienne, E.; Lutz-Wahl, S.; Fischer, L.; Kuzma, Marek; Pelantová, Helena; van Pelt, S.; Bolte, J.; Křen, Vladimír; Martínková, Ludmila

    2008-01-01

    Roč. 50, 2-4 (2008), s. 107-113 ISSN 1381-1177 R&D Projects: GA ČR GA203/05/2267; GA MŠk(CZ) LC06010; GA MŠk OC 171 Grant - others:XE(XE) ESF COST D25/0002/02; CZ(CZ) D10-CZ25/06-07; CZ(CZ) D-25 Institutional research plan: CEZ:AV0Z50200510 Keywords : rhodococcus erythropolis * nitrile hydratase * amidase Subject RIV: EE - Microbiology, Virology Impact factor: 2.015, year: 2008

  6. METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

    Energy Technology Data Exchange (ETDEWEB)

    John J. Kilbane III

    2003-12-01

    The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project will focus on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate amidase. The objective of the final phase of the project will be to develop derivative CN bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. The project is on schedule and no major difficulties have been encountered. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments have resulted in the isolation of promising cultures that may be capable of cleaving C-N bonds in aromatic amides, several amidase genes have been cloned and are currently undergoing directed evolution to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. Future research will address expression of these genes in Rhodococcus erythropolis. Enrichment culture experiments and directed evolution experiments continue to be a main focus of research activity and further work is required to obtain an appropriate amidase that will selectively cleave C-N bonds in aromatic substrates. Once an appropriate amidase gene is obtained it must be combined with genes encoding an enzyme capable of converting carbazole to 2'aminobiphenyl-2,3-diol: specifically carA genes. The carA genes from two sources have been cloned and are ready for construction of C-N bond cleavage

  7. Hydrolysis of Ibuprofen Nitrile and Ibuprofen Amide and Deracemisation of Ibuprofen Using Nocardia corallina B-276

    OpenAIRE

    Myrna Solís-Oba; Norberto Manjarrez; Aida Solís; Ricardo Lievano; Herminia Inés Pérez

    2012-01-01

    A novel application of whole cells of Nocardia corallina B-276 for the deracemisation of ibuprofen is reported. This microorganism successfully hydrolysed ibuprofen nitrile to ibuprofen amide, and ibuprofen amide to ibuprofen, using a suspension of cells in a potassium phosphate buffer solution (0.1 M, pH = 7.0). These results can be explained by the presence of NHase and amidase enzymes, but the reactions are not enantioselective and low ee values were obtained. However, (R)-ibuprofen was is...

  8. Biochemical response of Anticarsia gemmatalis fed with soybean plants pulverized with the synthetic trypsin inhibitor benzamidine

    International Nuclear Information System (INIS)

    Oliveira, M.G.A.; Pilon, A.M.; Pilon, F.M.; Ribeiro, F.R.; Silva, F.C.; Ribon, A.O.B.; Reis, A.P.; Visotto, L.E.; Guedes, R.N.C.; Oliveira, J.A.

    2008-01-01

    Full text: Insects are responsible for severe crop losses. New alternatives for pest control other than agrochemicals have been investigated. Protease inhibitors are one of the prime candidates effective against insect pests. In this work we studied the effect of the synthetic trypsin inhibitor benzamidine on the development of Anticarsia gemmatalis, an important pest of the soybean culture. Larvae were reared on soybean plants containing 0.00, 0.15, 0.30, 0.45, 0.60 and 0.75% (w/w) of benzamidine. After 6, 12, 24 and 48 h of feeding midgut extracts were prepared and assayed for enzymatic activity (proteolytic, amidasic and stearic). Benzamidine altered the activity patterns but was not able to totally abolish enzyme activity. The proteolytic, amidasic and stearic activity showed the higher time of inhibition in 48 h in concentration of 0,75%, the inhibition was the around 93%, 63.1% and 36.6%, respectively. We suggest that the presence of inhibitor has made insects to adapt and produce proteases which are insensitive to the action of benzamidine. (author)

  9. Biochemical response of Anticarsia gemmatalis fed with soybean plants pulverized with the synthetic trypsin inhibitor benzamidine

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, M.G.A.; Pilon, A.M.; Pilon, F.M.; Ribeiro, F.R.; Silva, F.C.; Ribon, A.O.B.; Reis, A.P.; Visotto, L.E. [Universidade Federal de Vicosa (UFV), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Biologia Molecular; Guedes, R.N.C. [Universidade Federal de Vicosa (UFV), Belo Horizonte, MG (Brazil). Dept. de Biologia Animal; Oliveira, J.A. [Universidade Federal de Vicosa (UFV), Belo Horizonte, MG (Brazil). Dept. de Quimica

    2008-07-01

    Full text: Insects are responsible for severe crop losses. New alternatives for pest control other than agrochemicals have been investigated. Protease inhibitors are one of the prime candidates effective against insect pests. In this work we studied the effect of the synthetic trypsin inhibitor benzamidine on the development of Anticarsia gemmatalis, an important pest of the soybean culture. Larvae were reared on soybean plants containing 0.00, 0.15, 0.30, 0.45, 0.60 and 0.75% (w/w) of benzamidine. After 6, 12, 24 and 48 h of feeding midgut extracts were prepared and assayed for enzymatic activity (proteolytic, amidasic and stearic). Benzamidine altered the activity patterns but was not able to totally abolish enzyme activity. The proteolytic, amidasic and stearic activity showed the higher time of inhibition in 48 h in concentration of 0,75%, the inhibition was the around 93%, 63.1% and 36.6%, respectively. We suggest that the presence of inhibitor has made insects to adapt and produce proteases which are insensitive to the action of benzamidine. (author)

  10. Thermophilic archaeal enzymes and applications in biocatalysis.

    Science.gov (United States)

    Littlechild, Jennifer A

    2011-01-01

    Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

  11. Molecular and functional characterization of peptidoglycan-recognition protein SC2 (PGRP-SC2) from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae.

    Science.gov (United States)

    Gan, Zhen; Chen, Shannan; Hou, Jing; Huo, Huijun; Zhang, Xiaolin; Ruan, Baiye; Laghari, Zubair Ahmed; Li, Li; Lu, Yishan; Nie, Pin

    2016-07-01

    PGRP-SC2, the member of PGRP family, plays an important role in regulation of innate immune response. In this paper, a PGRP-SC2 gene of Nile tilapia, Oreochromis niloticus (designated as On-PGRP-SC2) was cloned and its expression pattern under the infection of Streptococcus agalactiae was investigated. Sequence analysis showed main structural features required for amidase activity were detected in the deduced amino acid sequence of On-PGRP-SC2. In healthy tilapia, the On-PGRP-SC2 transcripts could be detected in all the examined tissues, with the most abundant expression in the muscle. When infected with S. agalactiae, there was a clear time-dependent expression pattern of On-PGRP-SC2 in the spleen, head kidney and brain. The assays for the amidase activity suggested that recombinant On-PGRP-SC2 protein had a Zn(2+)-dependent PGN-degrading activity. Moreover, our works showed that recombinant On-PGRP-SC2 protein could significantly reduce bacterial load in target organs attacked by S. agalactiae. These findings indicated that On-PGRP-SC2 may play important roles in the immune response to S. agalactiae in Nile tilapia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Molecular cloning and functional characterization of peptidoglycan recognition protein OmPGRP-L2 from the rainbow trout, Oncorhynchus mykiss.

    Science.gov (United States)

    Jang, Ju Hye; Kim, Hyun; Cho, Ju Hyun

    2017-10-01

    Peptidoglycan recognition proteins (PGRPs), a group of pattern recognition receptors (PRRs), are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. In teleost fish, several PGRPs have been characterized recently. They have both amidase activity and bactericidal activity and are involved in indirectly killing bacteria and regulating multiple signaling pathways. However, the knowledge of functional similarity and divergence between PGRP paralogs for their role as an immune modulator in teleost fish is still limited. In this study, we identified a novel PGRP paralog, termed OmPGRP-L2 from the rainbow trout (Oncorhynchus mykiss). OmPGRP-L2 contains the conserved PGRP domain and the four Zn 2+ -binding amino acid residues required for amidase activity. Quantitative RT-PCR analysis indicated that OmPGRP-L2 is highly expressed in liver. Overexpression of OmPGRP-L2 in a rainbow trout hepatocyte cell line RTH-149 challenged with Edwardsiella tarda resulted in down-regulation of IL-1β and TNF-α expression. When overexpressed in RTH-149 cells, OmPGRP-L2 inhibited NF-κB activity with or without bacterial stimulation. Collectively, these findings suggest that OmPGRP-L2 has an immunomodulatory function, via NF-κB inhibition in liver. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid.

    Directory of Open Access Journals (Sweden)

    Pradeep Mishra

    Full Text Available Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S-amide to (S-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH. IaaH is known to catalyse conversion of indole-3-acetamide (IAM to indole-3-acetic acid (IAA, which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To

  14. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid.

    Science.gov (United States)

    Mishra, Pradeep; Kaur, Suneet; Sharma, Amar Nath; Jolly, Ravinder S

    2016-01-01

    Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of

  15. Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo).

    Science.gov (United States)

    Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A

    2012-09-15

    This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After

  16. Distribution, industrial applications, and enzymatic synthesis of D-amino acids.

    Science.gov (United States)

    Gao, Xiuzhen; Ma, Qinyuan; Zhu, Hailiang

    2015-04-01

    D-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, D-amino acids have recently received more and more attention. Enzymes including D-hydantoinase, N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, D-peptidase, L-amino acid oxidase, D-amino acid aminotransferase, and D-amino acid dehydrogenase can be used for D-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, D-amino acid dehydrogenase method not only produces D-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention.

  17. Deciphering Physiological Functions of AHL Quorum Quenching Acylases

    Directory of Open Access Journals (Sweden)

    Putri D. Utari

    2017-06-01

    Full Text Available N-Acylhomoserine lactone (AHL-acylase (also known as amidase or amidohydrolase is a class of enzyme that belongs to the Ntn-hydrolase superfamily. As the name implies, AHL-acylases are capable of hydrolysing AHLs, the most studied signaling molecules for quorum sensing in Gram-negative bacteria. Enzymatic degradation of AHLs can be beneficial in attenuating bacterial virulence, which can be exploited as a novel approach to fight infection of human pathogens, phytopathogens or aquaculture-related contaminations. Numerous acylases from both prokaryotic and eukaryotic sources have been characterized and tested for the interference of quorum sensing-regulated functions. The existence of AHL-acylases in a multitude of organisms from various ecological niches, raises the question of what the physiological roles of AHL-acylases actually are. In this review, we attempt to bring together recent studies to extend our understanding of the biological functions of these enzymes in nature.

  18. A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry.

    Science.gov (United States)

    He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li

    2011-02-01

    Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

  19. [Transformation of 2- and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria].

    Science.gov (United States)

    Maksimova, Iu G; Vasil'ev, D M; Ovechkina, G V; Maksimov, A Iu; Demakov, V A

    2013-01-01

    The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt 1 strain possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 strain containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5-4 times lower compared to 4-cyanopyridine and 1.6-2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt 1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on china clay was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carbonic acids.

  20. Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout oncorhynchus-mykiss

    DEFF Research Database (Denmark)

    Kristjansson, Magnus M.; Nielsen, Henrik Hauch

    1992-01-01

    Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The approximate molecular weights of chymotrypsin I and II...... unstable at pH values below 5. The amidase activity of both enzymes increased with temperature up to about 55.degree. C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by strong calcium binding of the former. The trout chymotrypsins were significantly...... more active than bovine .alpha.-chymotrypsin when assayed against Suc-AAPF-NA at 25.degree. C and casein at low temperatures (10-20.degree. C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish....

  1. Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

    Science.gov (United States)

    Cibik, R; Chapot-Chartier, M P

    2000-11-01

    The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

  2. Identification of NH4+-regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis.

    Science.gov (United States)

    Schwab, Stefan; Ramos, Humberto J; Souza, Emanuel M; Pedrosa, Fábio O; Yates, Marshall G; Chubatsu, Leda S; Rigo, Liu U

    2007-05-01

    Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.

  3. A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

    DEFF Research Database (Denmark)

    Lappann, M.; Claus, H.; van Alen, T.

    2010-01-01

    formation, whereas biofilm formation of cc with low point prevalence (ST-8 cc and ST-11 cc) was eDNA-independent. For initial biofilm formation, a ST-32 cc type strain, but not a ST-11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N......-acetylmuramyl-l-alanine amidase genes. In late biofilms, outer membrane phospholipase A-dependent autolysis, which was observed in most cc, but not in ST-8 and ST-11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an e....... On the contrary, spreaders (ST-11 and ST-8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates....

  4. The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

    Science.gov (United States)

    Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

    2017-07-15

    Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

  5. Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

    Science.gov (United States)

    Lee, Jun Beom; Byeon, Jin Hee; Jang, Ho Am; Kim, Jiyeun Kate; Yoo, Jin Wook; Kikuchi, Yoshitomo; Lee, Bok Luel

    2015-09-14

    We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Effects of acidity and alkalinity on the stability of amidohydrolases in freshwater

    Energy Technology Data Exchange (ETDEWEB)

    Frankenberger, W.T. Jr.; Page, A.L.

    1983-01-01

    The influence of acidity and alkalinity on the enzymatic hydrolysis of amide-N in the aquatic system was investigated by determining the stability of L-asparaginase (EC (Enzyme Commission) 3.5.1.1), L-glutaminase (EC 3.5.1.2), and amidase (EC 3.5.1.4), and urease (EC 3.5.1.5) with respect to pH in three freshwater lakes. The water samples were first incubated at the indicated buffer pH (1-13) for 24 h; then each enzyme was assayed at its optimal pH (pH/sub opt/) under standardized conditions. L-Asparaginase retained essentially 100% of its activity within the pH range of 6-7 in Lake Evans and Lake Matthews, and 5-7 in Lake Perris. L-Glutaminase was active between pH 6 and 7 in the water samples surveyed. Amidase remained completely active at pH ranges of 4-8 in Lake Evans, 4-7 in Lake Matthews, and 5-8 in Lake Perris. Urease remained active over a broad pH range of 5-9 in Lake Evans, 5-8 in Lake Matthews, and 4-8 in Lake Perris. There was a progressive loss in enzyme activity below and above these pH ranges. The results suggest that the decline in aquatic amidohydrolase activities in relation to the pH-profile near their pH/sub opt/ was caused by a reversible reaction involving the ionization or deionization of functional groups of the active center of the protein, but under highly acidic or alkaline conditions (pH <4 to >9) the reduced activitiy appears to be due to irreversible inactivation of the enzyme.

  7. Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers

    Science.gov (United States)

    Carrera, Mónica; Böhme, Karola; Gallardo, José M.; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

    2017-01-01

    In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs. PMID:29312172

  8. PGRP-LB homolog acts as a negative modulator of immunity in maintaining the gut-microbe symbiosis of red palm weevil, Rhynchophorus ferrugineus Olivier.

    Science.gov (United States)

    Dawadi, Bishnu; Wang, Xinghong; Xiao, Rong; Muhammad, Abrar; Hou, Youming; Shi, Zhanghong

    2018-09-01

    Many notorious insect pests live in the symbiotic associations with gut microbiota. However, the mechanisms underlying how they host their gut microbiota are unknown. Most gut bacteria can release peptidoglycan (PGN) which is an important antigen to activate the immune response. Therefore, how to keep the appropriate gut immune intensity to host commensals while to efficiently remove enteropathogens is vital for insect health. This study is aimed at elucidating the roles of an amidase PGRP, Rf PGRP-LB, in maintaining the gut-microbe symbiosis of Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier. RfPGRP-LB is a secreted protein containing a typical PGRP domain. The existence of five conservative amino acid residues, being required for amidase activity, showed that RfPGRP-LB is a catalytic protein. Expression analysis revealed abundance of RfPGRP-LB transcripts in gut was dramatically higher than those in other tissues. RfPGRP-LB could be significantly induced against the infection of Escherichia coli. In vitro assays revealed that rRfPGRP-LB impaired the growth of E. coli and agglutinated bacteria cells obviously, suggesting RfPGRP-LB is a pathogen recognition receptor and bactericidal molecule. RfPGRP-LB knockdown reduced the persistence of E. coli in gut and load of indigenous gut microbiota significantly. Furthermore, the community structure of indigenous gut microbiota was also intensively altered by RfPGRP-LB silence. Higher levels of the antimicrobial peptide, attacin, were detected in guts of RfPGRP-LB silenced larvae than controls. Collectively, RfPGRP-LB plays multiple roles in modulating the homeostasis of RPW gut microbiota not only by acting as a negative regulator of mucosal immunity through PGN degradation but also as a bactericidal effector to prevent overgrowth of commensals and persistence of noncommensals. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Nitrile Metabolizing Yeasts

    Science.gov (United States)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  10. Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability.

    Directory of Open Access Journals (Sweden)

    Aurelia Delaune

    Full Text Available The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

  11. Molecular characterization of podoviral bacteriophages virulent for Clostridium perfringens and their comparison with members of the Picovirinae.

    Directory of Open Access Journals (Sweden)

    Nikolay V Volozhantsev

    Full Text Available Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

  12. Production of enantiomerically pure D-Phenylglycine using Pseudomonas aeruginosa 10145 as biocatalyst

    Directory of Open Access Journals (Sweden)

    F. O. M. Alonso

    2008-03-01

    Full Text Available Different bacterial strains were screened to detect nitrilase and/or nitrile hidratase/amidase activities towards benzonitrile, to be used as biocatalyst to produce enantiomerically pure non-proteinogenic amino acids using amino nitriles as starting material. The best biocatalyst found was Pseudomonas aeruginosa 10145, which showed high enzyme activities. Whole cells were used as catalyst for the transformation of 2-phenyl-2-amino-acetonitrile for the corresponding D-phenylglycine. The percentage conversion was followed by chiral HPLC. After 1 hour reaction 18% of 2-phenyl-2-amino-acetonitrile was converted into D-phenylglycine with an enantiomeric excess of over 95%. When an inducer was added to the media, an increase in nitrile hydrolyzing activities was detected, hence leading to total conversion of (R-2-phenyl-2-amino-acetonitrile to the corresponding amino acid in 30 min reaction. The isolated yield of the target product was 50% and its characterization was performed by polarimetry, chiral HPLC, IR-FT spectroscopy and GC-MS.

  13. Cell lysis and superoxide dismutase activities of highly radioresistant bacteria

    International Nuclear Information System (INIS)

    Yoshinaka, Taeko; Yano, Keiji; Yamaguchi, Hikoyuki

    1976-01-01

    The highly radioresistant bacterium, Arthrobacter radiotolerans, has been isolated from the radioactive hot spring of Misasa, and it does not sporulate, it is Gram-positive, and its color is pink to red. This bacterium shows the highest resistance to gamma-ray among Gram-positive resistants, but the lytic enzyme capable of lysing the cells of strong radioresistants and the surface structure of the cells are little known except those about Micrococcus radiodurans. The cells of the M. radiodurans can be lysed by Achramobacter lyticus enzyme, and electron microscopic observation and chemical analysis revealed the mutilayered surface structure of the cells consisting of an inner membrane, a mucopeptide wall layer and a very outer layer. The superoxide dismutase (SOD) activity of aerobic and anaerobic bacteria was studied, and the relatively high SOD activity of the M. radiodurans was found. The SOD function acts against the threat posed by the reactive superoxide radical being generated biologically, photochemically and radiochemically in the presence of molecular oxygen. In this paper, it is reported that the lytic enzyme No.2 obtained from Cytophaga sp., containing N-acetyl-muramyl-L-alanine amidase, peptidase and endopeptidase, and showing broad lytic spectra, was able to lyse the cells of A. radiotolerans and four radioresistant micrococci, and the radioresistant bacteria showed relatively high SOD activity except M. sp. 248. It is well known that superoxide anions are generated by aerobic irradiation, and are toxic to microbial cells. (Kako, I.)

  14. Cell lysis and superoxide dismutase activities of highly radioresistant bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Yoshinaka, T; Yano, K; Yamaguchi, H [Tokyo Univ. (Japan). Faculty of Agriculture

    1976-01-01

    The highly radioresistant bacterium, Arthrobacter radiotolerans, has been isolated from the radioactive hot spring of Misasa, and it does not sporulate, it is Gram-positive, and its color is pink to red. This bacterium shows the highest resistance to gamma-ray among Gram-positive resistants, but the lytic enzyme capable of lysing the cells of strong radioresistants and the surface structure of the cells are little known except those about Micrococcus radiodurans. The cells of the M. radiodurans can be lysed by Achramobacter lyticus enzyme, and electron microscopic observation and chemical analysis revealed the mutilayered surface structure of the cells consisting of an inner membrane, a mucopeptide wall layer and a very outer layer. The superoxide dismutase (SOD) activity of aerobic and anaerobic bacteria was studied, and the relatively high SOD activity of the M. radiodurans was found. The SOD function acts against the threat posed by the reactive superoxide radical being generated biologically, photochemically and radiochemically in the presence of molecular oxygen. In this paper, it is reported that the lytic enzyme No.2 obtained from Cytophaga sp., containing N-acetyl-muramyl-L-alanine amidase, peptidase and endopeptidase, and showing broad lytic spectra, was able to lyse the cells of A. radiotolerans and four radioresistant micrococci, and the radioresistant bacteria showedrelatively high SOD activity except M. sp. 248. It is well known that superoxide anions are generated by aerobic irradiation, and are toxic to microbial cells.

  15. Study of biodegradation of partially hydrolyzed polyacrylamide in an oil reservoir after polymer flooding

    International Nuclear Information System (INIS)

    Bao, M.; Chen, Q.; Li, Y.; Jiang, G.

    2009-01-01

    Studies have demonstrated that the amide group of polyacrylamides can provide a nitrogen source for microorganisms. However, the carbon backbone of the polymers cannot be cleaved by microbial activity. This study examined the biodegradability of partially hydrolyzed polyacrylamide (HPAM) in an aerobic environment both before and after bacterial biodegradation. Results of the infrared spectrum study indicated that the amide group of HPAM in the products was converted to a carboxyl group. High performance liquid chromatography analyses did not demonstrate the presence of acrylamide monomers. A scanning electron microscopy (SEM) study showed that the surfaces of HPAM particles had been altered by the biodegradation process. Results of the study indicated that the HPAM carbon backbone was metabolized by the bacteria during the course of its growth. It was hypothesized that the HPAM was initially utilized by the bacteria as a nitrogen source by the hydrolysis of the HPAM amide groups using an amidase enzyme. Oxidation of the carbon backbone chain then occurred by monooxygenase catalysis. It was concluded that the HPAM carbon backbone then served as a source for further bacterial growth and metabolism. 13 refs., 5 figs

  16. BioGPS descriptors for rational engineering of enzyme promiscuity and structure based bioinformatic analysis.

    Directory of Open Access Journals (Sweden)

    Valerio Ferrario

    Full Text Available A new bioinformatic methodology was developed founded on the Unsupervised Pattern Cognition Analysis of GRID-based BioGPS descriptors (Global Positioning System in Biological Space. The procedure relies entirely on three-dimensional structure analysis of enzymes and does not stem from sequence or structure alignment. The BioGPS descriptors account for chemical, geometrical and physical-chemical features of enzymes and are able to describe comprehensively the active site of enzymes in terms of "pre-organized environment" able to stabilize the transition state of a given reaction. The efficiency of this new bioinformatic strategy was demonstrated by the consistent clustering of four different Ser hydrolases classes, which are characterized by the same active site organization but able to catalyze different reactions. The method was validated by considering, as a case study, the engineering of amidase activity into the scaffold of a lipase. The BioGPS tool predicted correctly the properties of lipase variants, as demonstrated by the projection of mutants inside the BioGPS "roadmap".

  17. Cloning, characterization and effect of TmPGRP-LE gene silencing on survival of Tenebrio molitor against Listeria monocytogenes infection.

    Science.gov (United States)

    Tindwa, Hamisi; Patnaik, Bharat Bhusan; Kim, Dong Hyun; Mun, Seulgi; Jo, Yong Hun; Lee, Bok Luel; Lee, Yong Seok; Kim, Nam Jung; Han, Yeon Soo

    2013-11-14

    Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

  18. Cloning, Characterization and Effect of TmPGRP-LE Gene Silencing on Survival of Tenebrio Molitor against Listeria monocytogenes Infection

    Directory of Open Access Journals (Sweden)

    Yeon Soo Han

    2013-11-01

    Full Text Available Peptidoglycan recognition proteins (PGRPs are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP-type peptidoglycan to activate both the immune deficiency (Imd and proPhenoloxidase (proPO pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

  19. A Personal Retrospective: Elevating Anandamide (AEA by Targeting Fatty Acid Amide Hydrolase (FAAH and the Fatty Acid Binding Proteins (FABPs

    Directory of Open Access Journals (Sweden)

    Dale Deutsch

    2016-10-01

    Full Text Available This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and, until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and, as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that solubilize anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions.

  20. Decoding the biosynthesis and function of diphthamide, an enigmatic modification of translation elongation factor 2 (EF2

    Directory of Open Access Journals (Sweden)

    Raffael Schaffrath

    2014-05-01

    Full Text Available Diphthamide is a highly conserved modification of archaeal and eukaryal translation elongation factor 2 (EF2 and yet why cells need EF2 to contain diphthamide is unclear. In yeast, the first steps of diphthamide synthesis and the genes (DPH1-DPH5 required to form the intermediate diphthine are well-documented. However, the last step, amidadation of diphthine to diphthamide, had largely been ill-defined. Remarkably, through mining genome-wide synthetic gene array (SGA and chemical genomics databases, recent studies by Uthman et al. [PLoS Genetics (2013 9, e1003334] and Su et al. [Proc. Natl. Acad. Sci. USA (2012 109, 19983-19987] have identified two more diphthamide players, DPH6 and DPH7. Consistent with roles in the amidation step, dph6 and dph7 deletion strains fail to complete diphthamide synthesis and accumulate diphthine-modified EF2. In contrast to Dph6, the catalytically relevant amidase, Dph7 appears to be regulatory. As shown by Uthman et al., it promotes dissociation of diphthine synthase (Dph5 from EF2, allowing diphthine amidation by Dph6 to occur and thereby coupling diphthine synthesis to the terminal step in the pathway. Remarkably, the study by Uthman et al. suggests that Dph5 has a novel role as an EF2 inhibitor that affects cell growth when diphthamide synthesis is blocked or incomplete and, importantly, shows that diphthamide promotes the accuracy of EF2 performance during translation.

  1. Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides.

    Science.gov (United States)

    Krstanović, Marina; Frkanec, Ruza; Vranesić, Branka; Ljevaković, Durdica; Sporec, Vesna; Tomasić, Jelka

    2002-06-25

    The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.

  2. Pharmacological characterization of hydrolysis-resistant analogs of oleoylethanolamide with potent anorexiant properties.

    Science.gov (United States)

    Astarita, Giuseppe; Di Giacomo, Barbara; Gaetani, Silvana; Oveisi, Fariba; Compton, Timothy R; Rivara, Silvia; Tarzia, Giorgio; Mor, Marco; Piomelli, Daniele

    2006-08-01

    Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-alpha (PPAR-alpha). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-alpha with high potency in vitro, and persistently reduce feeding when administered in vivo either parenterally or orally. The most potent of these compounds, (Z)-(R)-9-octadecenamide,N-(2-hydroxyethyl,1-methyl) (KDS-5104), stimulates transcriptional activity of PPAR-alpha with a half-maximal effective concentration (EC50) of 100 +/- 21 nM (n = 11). Parenteral administration of KDS-5104 in rats produces persistent dose-dependent prolongation of feeding latency and postmeal interval (half-maximal effective dose, ED50 = 2.4 +/- 1.8 mg kg(-1) i.p.; n = 18), as well as increased and protracted tissue exposure compared with OEA. Oral administration of the compound also results in a significant tissue exposure and reduction of food intake in free-feeding rats. These results suggest that the endogenous high-affinity PPAR-alpha agonist OEA may provide a scaffold for the discovery of novel orally active PPAR-alpha ligands.

  3. Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Jeffrey L Bose

    Full Text Available The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM and a glucosaminidase (GL. Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

  4. Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

    Science.gov (United States)

    Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

    2018-04-01

    Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

  5. Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

    Science.gov (United States)

    Bose, Jeffrey L; Lehman, McKenzie K; Fey, Paul D; Bayles, Kenneth W

    2012-01-01

    The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

  6. Real-time PCR detection of Fe-type nitrile hydratase genes from environmental isolates suggests horizontal gene transfer between multiple genera.

    Science.gov (United States)

    Coffey, Lee; Owens, Erica; Tambling, Karen; O'Neill, David; O'Connor, Laura; O'Reilly, Catherine

    2010-11-01

    Nitriles are widespread in the environment as a result of biological and industrial activity. Nitrile hydratases catalyse the hydration of nitriles to the corresponding amide and are often associated with amidases, which catalyze the conversion of amides to the corresponding acids. Nitrile hydratases have potential as biocatalysts in bioremediation and biotransformation applications, and several successful examples demonstrate the advantages. In this work a real-time PCR assay was designed for the detection of Fe-type nitrile hydratase genes from environmental isolates purified from nitrile-enriched soils and seaweeds. Specific PCR primers were also designed for amplification and sequencing of the genes. Identical or highly homologous nitrile hydratase genes were detected from isolates of numerous genera from geographically diverse sites, as were numerous novel genes. The genes were also detected from isolates of genera not previously reported to harbour nitrile hydratases. The results provide further evidence that many bacteria have acquired the genes via horizontal gene transfer. The real-time PCR assay should prove useful in searching for nitrile hydratases that could have novel substrate specificities and therefore potential in industrial applications.

  7. Enzymatic degradation of aliphatic nitriles by Rhodococcus rhodochrous BX2, a versatile nitrile-degrading bacterium.

    Science.gov (United States)

    Fang, Shumei; An, Xuejiao; Liu, Hongyuan; Cheng, Yi; Hou, Ning; Feng, Lu; Huang, Xinning; Li, Chunyan

    2015-06-01

    Nitriles are common environmental pollutants, and their removal has attracted increasing attention. Microbial degradation is considered to be the most acceptable method for removal. In this work, we investigated the biodegradation of three aliphatic nitriles (acetonitrile, acrylonitrile and crotononitrile) by Rhodococcus rhodochrous BX2 and the expression of their corresponding metabolic enzymes. This organism can utilize all three aliphatic nitriles as sole carbon and nitrogen sources, resulting in the complete degradation of these compounds. The degradation kinetics were described using a first-order model. The degradation efficiency was ranked according to t1/2 as follows: acetonitrile>trans-crotononitrile>acrylonitrile>cis-crotononitrile. Only ammonia accumulated following the three nitriles degradation, while amides and carboxylic acids were transient and disappeared by the end of the assay. mRNA expression and enzyme activity indicated that the tested aliphatic nitriles were degraded via both the inducible NHase/amidase and the constitutive nitrilase pathways, with the former most likely preferred. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. [Applications of nitrile converting enzymes in the production of fine chemicals].

    Science.gov (United States)

    Zheng, Yuguo; Xue, Yaping; Liu, Zhiqiang; Zheng, Renchao; Shen, Yinchu

    2009-12-01

    Nitriles are an important type of synthetic intermediates in the production of fine chemicals because of their easy preparations and versatile transformations. The traditional chemical conversion of nitriles to carboxylic acids and amides is feasible but it requires relatively harsh conditions of heat, acid or alkali. Nitrile converting enzymes (nitrilase, nitrile hydratase and amidase) which are used as biocatalyst for the production of fine chemicals have attracted substantial interest because of their ability to convert readily available nitriles into the corresponding higher value amides or acids under mild conditions with excellent chemo-, regio- and stereo-selectivities. Many nitrile converting enzymes have been explored and widely used for the production of fine chemicals. In this paper, various examples of biocatalytic synthesis of pharmaceuticals and their intermediates, agrochemicals and their intermediates, food and feed additives, and other fine chemicals are presented. In the near future, an increasing number of novel nitrile converting enzymes will be screened and their potential in the production of useful fine chemicals will be further exploited.

  9. Asparagine and glycine metabolism in rat liver mitochondria and in mouse L5178Y lymphoma cells resistant or sensitive to the anticancer drug L-asparaginase

    Energy Technology Data Exchange (ETDEWEB)

    Keefer, J.F. Jr.

    1986-01-01

    Rat liver mitochondrial asparagine was found to be degraded via an aminotransferase and omega-amidase. Evidence includes oxaloacetate production from asparagine only when glyoxylate was added and production of radiolabeled ..cap alpha..-ketosuccinamate via metabolism of (U-/sup 14/C)asparagine. In the cytosol, asparagine is degraded primarily via asparaginase and subsequent transamination. A new HPLC technique for separation of citric acid cycle intermediates was developed using: ion pairing with 20 mM each to tetrabutylammonium hydroxide and Na/sub 2/SO/sub 4/; pH 7.0; isocratic elution; and detection at 210 nm. Amino acid content of mouse lymphoma cells either sensitive (L5178Y) or resistant (L5178Y/L-ASE) to the anticancer drug L-asparaginase was studied. The concentration of asparagine was 1.5 times higher and the concentrations of the essential amino acids histidine, methionine, valine and phenylalanine were two times higher in asparaginase-resistant than sensitive cells. In vivo but not in vitro studies indicated that glucine decreases in sensitive but not resistant cells upon asparaginase treatment. Asparagine and glycine metabolism was further studied using /sup 14/C radiolabel conversion of asparagine, glyoxylate, glycine and serine. Glycine metabolism is especially important in lymphomas and leukemias because these cells contain higher concentrations of glycine that other cancer and normal cells. Therefore, glycine levels were studied and were found to decrease in sensitive but not resistant cells upon asparaginase administration.

  10. Determination of radiochemical purity of 125I-TOC and 125I-F-PGA

    International Nuclear Information System (INIS)

    Yang Keya; Fan Wo; Zhang Youjiu; Xu Yujie; Zhu Ran; Hu Mingjiang

    2006-01-01

    To explore whether there is accordance among three determination methods of the radiochemical purity of [Tyr 3 ] octreotide (TOC) and folate-penicillin G amidase conjugate (F-PGA), which are both labeled with 125 I by Iodogen method, the RCP of the labelings are determined by high performance liquid chromatography (HPLC), paper chromatography and trichloroacetic acid (TCA) precipitation, in which four different concentrations of proteins are used to investigate the effect of them on the determination of RCP. It is shown that both HPLC and paper chromatography can separate the labelings from free iodine efficiently, though HPLC is the most precise and reliable method to determinate RCP of such labelings. In TCA precipitation, the RCP measured with 0.2%BSA is the lowest, but those with three other concentrations of the BSA are similar (P>0.05). When RCP 0.05), whereas higher than that with HPLC (P 10%, the RCP of 125 I-TOC obtained by TCA precipitation is a bit lower than those by two other methods (P 0.05), and there are no significant differences to determinate the RCP of 125 I-F-PGA (P>0.05). The three methods are correlated each other (r=0.0996-0.999, P<0.001). (authors)

  11. Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.).

    Science.gov (United States)

    Omar, S A; Abd-Alla, M H

    2000-03-01

    The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi. During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt). The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi. Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately. All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates. Four fungal species, Aspergillus awamori, A. flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation. The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion. Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity. Cu(1+), Fe3+ and Al3+ showed a stimulatory effect. Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities. Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%.

  12. Peptidoglycan Hydrolases of Escherichia coli

    Science.gov (United States)

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  13. The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk.

    Science.gov (United States)

    Sheng, Long; He, Zhenjiao; Chen, Jiahui; Liu, Yaofa; Ma, Meihu; Cai, Zhaoxia

    2017-03-01

    Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk.

    Science.gov (United States)

    Sheng, Long; He, Zhenjiao; Liu, Yaping; Ma, Meihu; Cai, Zhaoxia

    2018-03-01

    Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN 308 and ASN 409 ) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI-MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

    Science.gov (United States)

    Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

    2017-12-01

    Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

  16. Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

    Science.gov (United States)

    Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna

    2012-03-01

    Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.

  17. Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst

    Science.gov (United States)

    Liria, Cleber W.; Ungaro, Vitor A.; Fernandes, Raphaella M.; Costa, Natália J. S.; Marana, Sandro R.; Rossi, Liane M.; Machini, M. Teresa

    2014-11-01

    Enzyme-catalyzed chemical processes are selective, very productive, and generate little waste. Nevertheless, they may be optimized using enzymes bound to solid supports, which are particularly important for protease-mediated reactions since proteases undergo fast autolysis in solution. Magnetic nanoparticles are suitable supports for this purpose owing to their high specific surface area and to be easily separated from reaction media. Here we describe the immobilization of bovine α-chymotrypsin (αCT) on silica-coated superparamagnetic nanoparticles (Fe3O4@silica) and the characterization of the enzyme-nanoparticle hybrid (Fe3O4@silica-αCT) in terms of protein content, properties, recovery from reaction media, application, and reuse in enzyme-catalyzed peptide synthesis. The results revealed that (i) full acid hydrolysis of the immobilized protease followed by amino acid analysis of the hydrolyzate is a reliable method to determine immobilization yield; (ii) despite showing lower amidase activity and a lower K cat/ K m value for a specific substrate than free αCT, the immobilized enzyme is chemically and thermally more stable, magnetically recoverable from reaction media, and can be consecutively reused for ten cycles to catalyze the amide bond hydrolysis and ester hydrolysis of the protected dipeptide Z-Ala-Phe-OMe. Altogether, these properties indicate the potential of Fe3O4@silica-αCT to act as an efficient, suitably stable, and reusable catalyst in amino acid, peptide, and protein chemistry as well as in proteomic studies.

  18. Purification and Partial Characterization of a Novel Bacteriocin Synthesized by Lactobacillus paracasei HD1-7 Isolated from Chinese Sauerkraut Juice.

    Science.gov (United States)

    Ge, Jingping; Sun, Yanyang; Xin, Xing; Wang, Ying; Ping, Wenxiang

    2016-01-14

    Bacteriocins have antimicrobial activities against food-spoiling bacteria and food-borne pathogens. Paracin 1.7, a bacteriocin synthesized by Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut juice, was studied. Following partial purification with ammonium sulfate precipitation, CM Sepharose Fast Flow, and Sephadex G-10 chromatography, the molecular weight of Paracin 1.7 was about 10 kDa based on Tricine-SDS-PAGE results. A 2.87 fold purified bacteriocin was produced, reaching a final yield of 39.93% and the specific activity of 1.56 × 10(3) AU/mg. The N-terminal amino acid sequence of Paracin 1.7 was VSNTFFA, and the LC/LTQ results revealed that the N-terminal amino acid sequence was similar to that of ABC-type oligopeptide transport system protein and N-acetylmuramoyl-L-alanine amidase. Paracin 1.7 was sensitive to protease K, had antimicrobial activities at a broad pH range (3.0-8.0), and was heat resistant (121 °C for 20 min). Paracin 1.7 from Lactobacillus paracasei HD1-7 is a novel bacteriocin that has potential applications in food preservation.

  19. Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis.

    Directory of Open Access Journals (Sweden)

    Sanjiv Kumar

    Full Text Available Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA, Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase and Rv0309 (L,D-transpeptidase bind to fibronectin and laminin. We report Rv2599 (membrane protein, Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.

  20. Characterization, Genome Sequence, and Analysis of Escherichia Phage CICC 80001, a Bacteriophage Infecting an Efficient L-Aspartic Acid Producing Escherichia coli.

    Science.gov (United States)

    Xu, Youqiang; Ma, Yuyue; Yao, Su; Jiang, Zengyan; Pei, Jiangsen; Cheng, Chi

    2016-03-01

    Escherichia phage CICC 80001 was isolated from the bacteriophage contaminated medium of an Escherichia coli strain HY-05C (CICC 11022S) which could produce L-aspartic acid. The phage had a head diameter of 45-50 nm and a tail of about 10 nm. The one-step growth curve showed a latent period of 10 min and a rise period of about 20 min. The average burst size was about 198 phage particles per infected cell. Tests were conducted on the plaques, multiplicity of infection, and host range. The genome of CICC 80001 was sequenced with a length of 38,810 bp, and annotated. The key proteins leading to host-cell lysis were phylogenetically analyzed. One protein belonged to class II holin, and the other two belonged to the endopeptidase family and N-acetylmuramoyl-L-alanine amidase family, respectively. The genome showed the sequence identity of 82.7% with that of Enterobacteria phage T7, and carried ten unique open reading frames. The bacteriophage resistant E. coli strain designated CICC 11021S was breeding and its L-aspartase activity was 84.4% of that of CICC 11022S.

  1. Isolation and Characterization of Polyacrylamide-Degrading Bacteria from Dewatered Sludge

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    Feng Yu

    2015-04-01

    Full Text Available Polyacrylamide (PAM is a water-soluble polymer that is widely used as a flocculant in sewage treatment. The accumulation of PAM affects the formation of dewatered sludge and potentially produces hazardous monomers. In the present study, the bacterial strain HI47 was isolated from dewatered sludge. This strain could metabolize PAM as its sole nutrient source and was subsequently identified as Pseudomonas putida. The efficiency of PAM degradation was 31.1% in 7 days and exceeded 45% under optimum culture condition (pH 7.2, 39 °C and 100 rpm. The addition of yeast extract and glucose improved the bacterial growth and PAM degradation. The degraded PAM samples were analyzed by gel-filtration chromatography, Fourier transform infrared and high-performance liquid chromatography. The results showed that high-molecular-weight PAM was partly cleaved to small molecular oligomer derivatives and part of the amide groups of PAM had been converted to carboxyl groups. The biodegradation did not accumulate acrylamide monomers. Based on the SDS-PAGE and N-terminal sequencing results, the PAM amide groups were converted into carboxyl groups by a PAM-induced extracellular enzyme from the aliphatic amidase family.

  2. Microbial nitrilases: versatile, spiral forming, industrial enzymes.

    Science.gov (United States)

    Thuku, R N; Brady, D; Benedik, M J; Sewell, B T

    2009-03-01

    The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a alpha beta beta alpha-alpha beta beta alpha sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

  3. Enzymes in Fermented Fish.

    Science.gov (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  4. Degradation of the metal-cyano complex tetracyanonickelate (II) by Fusarium oxysporum N-10.

    Science.gov (United States)

    Yanase, H; Sakamoto, A; Okamoto, K; Kita, K; Sato, Y

    2000-03-01

    A fungus with the ability to utilize a metalcyano compound, tetracyanonickelate (II) ¿K2[Ni (CN)4]; TCN¿, as its sole source of nitrogen was isolated from soil and identified as Fusarium oxysporum N-10. Both intact mycelia and cell-free extract of the strain catalyzed hydrolysis of TCN to formate and ammonia and produced formamide as an intermediate, thereby indicating that a hydratase and an amidase sequentially participated in the degradation of TCN. The enzyme catalyzing the hydration of TCN was purified approximately ten-fold from the cell-free extract of strain N-10 with a yield of 29%. The molecular mass of the active enzyme was estimated to be 160 kDa. The enzyme appears to exist as a homotetramer, each subunit having a molecular mass of 40 kDa. The enzyme also catalyzed the hydration of KCN, with a cyanide-hydrating activity 2 x 10(4) times greater than for TCN. The kinetic parameters for TCN and KCN indicated that hydratase isolated from F. oxysporum was a cyanide hydratase able to utilize a broad range of cyano compounds and nitriles as substrates.

  5. Rimonabant, a selective cannabinoid1 receptor antagonist, protects against light-induced retinal degeneration in vitro and in vivo.

    Science.gov (United States)

    Imamura, Tomoyo; Tsuruma, Kazuhiro; Inoue, Yuki; Otsuka, Tomohiro; Ohno, Yuta; Ogami, Shiho; Yamane, Shinsaku; Shimazawa, Masamitsu; Hara, Hideaki

    2017-05-15

    The endocannabinoid system is involved in some neurodegenerative diseases such as Alzheimer's disease. An endogenous constellation of proteins related to cannabinoid 1 receptor signaling, including free fatty acids, diacylglycerol lipase, and N-acylethanolamine-hydrolyzing acid amidase, are localized in the murine retina. Moreover, the expression levels of endogenous agonists of cannabinoid receptors are changed in the vitreous fluid. However, the role of the endocannabinoid system in the retina, particularly in the light-induced photoreceptor degeneration, remains unknown. Therefore, we investigated involvement of the cannabinoid 1 receptor in light-induced retinal degeneration using in vitro and in vivo models. To evaluate the effect of cannabinoid 1 receptors in light irradiation-induced cell death, the mouse retinal cone-cell line (661W) was treated with a cannabinoid 1 receptor antagonist, rimonabant. Time-dependent changes of expression and localization of retinal cannabinoid 1 receptors were measured using Western blot and immunostaining. Retinal damage was induced in mice by exposure to light, followed by intravitreal injection of rimonabant. Electroretinograms and histologic analyses were performed. Rimonabant suppressed light-induced photoreceptor cell death. Cannabinoid 1 receptor expression was upregulated by light exposure. Treatment with rimonabant improved both a- and b-wave amplitudes and the thickness of the outer nuclear layer. These results suggest that the cannabinoid 1 receptor is involved in light-induced retinal degeneration and it may represent a therapeutic target in the light-induced photoreceptor degeneration related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Using directed evolution to probe the substrate specificity of mandelamide hydrolase.

    Science.gov (United States)

    Wang, Pan-Fen; Yep, Alejandra; Kenyon, George L; McLeish, Michael J

    2009-02-01

    Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliphatic substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homology model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly202 appears to control the preference for aromatic substrates as the G202A variant showed three orders of magnitude decrease in k(cat)/K(m) for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant.

  7. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

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    Steven M. Swift

    2015-06-01

    Full Text Available Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

  8. Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum

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    Neelamegan Dhamodharan

    2012-06-01

    Full Text Available Abstract Background Anandamide (Arachidonoyl ethanolamide is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH. In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide. Results A putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates. Conclusions This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.

  9. Plasmin enzymatic activity in the presence of actin

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    Yusova E. I.

    2015-10-01

    Full Text Available Aim. To study the changes in the plasmin activity towards substrates with high and low molecular mass in the presence of actin. Methods. The proteins used for this investigation were obtained by affinity chromatography and gel-filtration. The plasmin enzymatic activity was determined by a turbidimetric assay and a chromogenic substrate-based assay. The enzyme linked immunosorbent assay and biotin-avidin-phosphatase system were used to study the interaction of plasminogen and its fragments with actin. Results. It was shown that G-actin causes 1.5-fold decrease in the rate of polymeric fibrin hydrolysis by plasmin and Glu-plasminogen activated by the tissue plasminogen activator. However, actin did not impede plasmin autolysis and had no influence on its amidase activity. We have studied an interaction of biotinylated Glu-plasminogen and its fragments (kringle 1-3, kringle 4 and mini-plasminogen with immobilized G-actin. Glu-plasminogen and kringle 4 had a high affinity towards actin (C50 is 113 and 117 nM correspondingly. Mini-plasminogen and kringe 4 did not bind to actin. A similar affinity of Glu-plasminogen and kringle 1-3 towards actin proves the involvement of the kringle 1-3 lysine-binding sites of the native plasminogen form in the actin interaction. Conclusions. Actin can modulate plasmin specificity towards high molecular mass substrates through its interaction with lysine-binding sites of the enzyme kringle domains. Actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for thelysine binding sites of plasminogen/plasmin.

  10. Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

    International Nuclear Information System (INIS)

    Wang, Tianyu; Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng; Liu, Wei

    2013-01-01

    The structure of the Tse3–Tsi3 complex associated with the bacterial type VI secretion system of P. aeruginosa has been solved and refined at 1.9 Å resolution. The structural basis of the recognition of the muramidase effector and its inactivation by its cognate immunity protein is revealed. The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair

  11. [Nicotinamidase and the so-called pyrazinamidase in mycobacteria; the simultaneous occurrence of both activites (author's transl)].

    Science.gov (United States)

    Tarnok, I; Pechmann, H; Krallmann-Wenzel, U; Röhrscheidt, E; Tarnok, Z

    1979-07-01

    Nicotin- and the so-called pyrazinamidase (in the following: "pyrazinamidase") have been found in strains of four mycobacteria species, M. fortuitum, M. gastri, M. bovis and M. microti. These findings are in contradiction to those summarized in Bergey's Manual of Determinative Bacteriology (1974). The reason for the discrepancies is that the original method (Bönicke, 1961) for amidase determination has not taken the following aspects into consideration: a) The inducibility of the nicotin- and "pyrazinamidase" (example: M. fortuitum); b) The temperature sensitivity of these enzymes (M. gastri); c) The light sensitivity of nicotinamidase (in photochromogenic M. gastri strains); d) The optimal substrate concentration which must be at least 4 mM instead of 0,8 mm. The following consequences can be drawn for the taxonomy and biochemistry of the tested organisms: e) The species status of M. gastri should be annuled. The main difference between M. gastri and M. kansasii consists only of the non-agglutinability of M. gastri by anti-M. kansasii serum. "Pyrazinamidase" and also nitrate reductase (Tarnok et al., in press) are positive in strains of both species; f) M. bovis possesses nicotin- and "pyrazinamidase" as M. tuberculosis too. Thus, these two species are more closely related than suggested earlier; g) Till now, no Mycobacterium has been found showing nicotinamidase without "pyrazinamidase" activity (or vice versa). It seems to be very probable that nicotinamidase, an enzyme of low substrate specificity, is able to hydrolyze several compounds with a nicotinamide-like structure such as pyrazinamide. Thus, we suggest the annulment of the term pyrazinamidase or the employment of quotation marks ("pyrazinamidase") to show the fictitious value of this designation.

  12. Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides.

    Science.gov (United States)

    Boshoff, H I; Mizrahi, V

    2000-10-01

    A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.

  13. Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin.

    Science.gov (United States)

    Sharma, Meenakshi; Kumar, Dinesh; Poluri, Krishna Mohan

    2016-08-23

    Bacteriophages are the most abundant and diverse biological entities on earth. Bacteriophage endolysins are unique peptidoglycan hydrolases and have huge potential as effective enzybiotics in various infectious models. T7 bacteriophage endolysin (T7L), also known as N-acetylmuramoyl-l-alanine amidase or T7 lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the l-alanine of the peptidoglycan layer. Although the activity profiles of several of the T7 family members have been known for many years, the molecular basis for their pH-dependent differential activity is not clear. In this study, we explored the pH-induced structural, stability, and activity characteristics of T7L by applying a variety of biophysical techniques and protein nuclear magnetic resonance (NMR) spectroscopy. Our studies established a reversible structural transition of T7L below pH 6 and the formation of a partially denatured conformation at pH 3. This low-pH conformation is thermally stable and exposed its hydrophobic pockets. Further, NMR relaxation measurements and structural analysis unraveled that T7L is highly dynamic in its native state and a network of His residues are responsible for the observed pH-dependent conformational dynamics and transitions. As bacteriophage chimeric and engineered endolysins are being developed as novel therapeutics against multiple drug resistance pathogens, we believe that our results are of great help in designing these entities as broadband antimicrobial and/or antibacterial agents.

  14. Population pharmacokinetics of modafinil acid and estimation of the metabolic conversion of modafinil into modafinil acid in 5 major ethnic groups of China.

    Science.gov (United States)

    Wu, Ke-hua; Guo, Tao; Deng, Chen-hui; Guan, Zheng; Li, Liang; Zhou, Tian-yan; Lu, Wei

    2012-11-01

    To describe the population pharmacokinetic profile of modafinil acid and to compare the extent of metabolism of modafinil into modafinil acid in 5 major ethnic groups (Han, Mongolian, Korean, Uygur, and Hui) of China. In a multi-center, open-label, single dose clinical trial, 49 healthy volunteers from the 5 ethnic groups received 200 mg of modafinil orally. Blood samples for pharmacokinetic evaluation of modafinil and modafinil acid were drawn before and at different time after the administration. Systematic population pharmacokinetic (PopPK) modeling for modafinil acid was conducted, integrating with our previous PopPK model for modafinil. The influence of ethnicity, gender, height, body weight and body mass index (BMI) was estimated. The extent of metabolism of modafinil into modafinil acid, expressed as the relative conversion fraction, was estimated and compared among the 5 ethnic groups. When combined with the PopPK model of modafinil, the concentration of modafinil acid versus time profile was best described with a one-compartment model. The typical clearance and volume of distribution for modafinil acid were 4.94 (l/h) and 2.73 (l), respectively. The Korean group had 25% higher clearance, and the Uygur and Hui groups had 12% higher clearance than the Han group. The median for the relative conversion fraction was 0.53 for Koreans, and 0.24 for the other 4 ethnicities. Ethnicity has significant influence on the clearance of modafinil acid. When patients in the 5 ethnic groups are administered drugs or prodrugs catalyzed by esterases and/or amidases, the variability in the extent of drug metabolism should be considered.

  15. Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori.

    Science.gov (United States)

    Bury-Moné, Stéphanie; Thiberge, Jean-Michel; Contreras, Monica; Maitournam, Aboubakar; Labigne, Agnès; De Reuse, Hilde

    2004-07-01

    The virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts. Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model. Little is known about how H. pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer. Thus, we compared gene expression profiles of H. pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real-time polymerase chain reaction, dot-blots or enzymatic assays. During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated. We found that acidity is a signal modulating the expression of several virulence factors. Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage. Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC-HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry. Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H. pylori. Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal-responsive transcriptional regulators (NikR and Fur) and an essential two-component response regulator (HP0166, OmpR-like) as effectors of the H. pylori acid response. Finally, a nikR-fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen.

  16. Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tianyu [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng, E-mail: dcwang@ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Wei, E-mail: dcwang@ibp.ac.cn [The Third Military Medical University, Chongqing 400038 (China); Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2013-10-01

    The structure of the Tse3–Tsi3 complex associated with the bacterial type VI secretion system of P. aeruginosa has been solved and refined at 1.9 Å resolution. The structural basis of the recognition of the muramidase effector and its inactivation by its cognate immunity protein is revealed. The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair.

  17. The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood.

    Science.gov (United States)

    Oliveira, Ana; Leite, Marta; Kluskens, Leon D; Santos, Sílvio B; Melo, Luís D R; Azeredo, Joana

    2015-01-01

    Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

  18. Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Zhan, Ming-Yue; Yang, Pei-Jin; Rao, Xiang-Jun

    2018-02-01

    Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella. © 2017 Wiley Periodicals, Inc.

  19. Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

    Science.gov (United States)

    Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

    2018-01-01

    Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

  20. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

    Science.gov (United States)

    Deutsch, Dale G

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

  1. In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

    Science.gov (United States)

    Oechslin, Frank; Daraspe, Jean; Giddey, Marlyse; Moreillon, Philippe; Resch, Grégory

    2013-12-01

    Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

  2. N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

    Energy Technology Data Exchange (ETDEWEB)

    Clagett-Dame, M.; McKelvy, J.F. (Abbott Laboratories, Abbott Park, IL (USA))

    1989-10-01

    The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-(125I)iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide.

  3. N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

    International Nuclear Information System (INIS)

    Clagett-Dame, M.; McKelvy, J.F.

    1989-01-01

    The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide

  4. Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features.

    Science.gov (United States)

    Liljeblad, Arto; Kallio, Pauli; Vainio, Marita; Niemi, Jarmo; Kanerva, Liisa T

    2010-02-21

    Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper.

  5. Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

    Science.gov (United States)

    Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

    1984-03-01

    N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

  6. Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes.

    Science.gov (United States)

    Anantharaman, Vivek; Aravind, L

    2003-01-01

    Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-gamma-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages. Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include lecithin retinol acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.

  7. SpxB is a suicide gene of Streptococcus pneumoniae and confers a selective advantage in an in vivo competitive colonization model.

    Science.gov (United States)

    Regev-Yochay, Gili; Trzcinski, Krzysztof; Thompson, Claudette M; Lipsitch, Marc; Malley, Richard

    2007-09-01

    The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H(2)O(2)), not LytA, and that the gene responsible for H(2)O(2) production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H(2)O(2) in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing DeltaspxB mutants that do not produce H(2)O(2). Likewise, addition of H(2)O(2) to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While DeltalytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H(2)O(2) has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the DeltaspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.

  8. The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23 with High Potential to Control American Foulbrood.

    Directory of Open Access Journals (Sweden)

    Ana Oliveira

    Full Text Available Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23 encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0, showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

  9. Identification of a novel gene cluster in the upstream region of the S-layer gene sbpA involved in cell wall metabolism of Lysinibacillus sphaericus CCM 2177 and characterization of the recombinantly produced autolysin and pyruvyl transferase.

    Science.gov (United States)

    Pleschberger, Magdalena; Hildner, Florian; Rünzler, Dominik; Gelbmann, Nicola; Mayer, Harald F; Sleytr, Uwe B; Egelseer, Eva M

    2013-05-01

    The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

  10. A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.

    Science.gov (United States)

    Filatova, Lyubov Y; Donovan, David M; Ishnazarova, Nadiya T; Foster-Frey, Juli A; Becker, Stephen C; Pugachev, Vladimir G; Balabushevich, Nadezda G; Dmitrieva, Natalia F; Klyachko, Natalia L

    2016-10-01

    A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal ( opt ) conditions: pH opt 6.0-10.0, t opt 20-30 °C, NaCl opt 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

  11. Understanding the Role of Host Hemocytes in a Squid/Vibrio Symbiosis Using Transcriptomics and Proteomics

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    Andrew J. Collins

    2012-05-01

    Full Text Available The symbiosis between the squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, serves as a model for understanding interactions between beneficial bacteria and animal hosts. The establishment and maintenance of the association is highly specific and depends on the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environment. Current evidence suggests that the host’s cellular innate immune system, in the form of macrophage-like hemocytes, helps to mediate host tolerance of V. fischeri. To begin to understand the role of hemocytes in this association, we analyzed these cells by high-throughput 454 transcriptomic and liquid chromatography/ tandem mass spectrometry (LC-MS/MS proteomic analyses. 454 high-throughput sequencing produced 650,686 reads totaling 279.9 Mb while LC-MS/MS analyses of circulating hemocytes putatively identified 702 unique proteins. Several receptors involved with the recognition of microbial associated molecular patterns (MAMPs were identified. Among these was a complete open reading frame (ORF to a putative peptidoglycan recognition protein (EsPGRP5 that has conserved residues for amidase activity. Assembly of the hemocyte transcriptome showed EsPGRP5 had high coverage, suggesting it is among the 5% most abundant transcripts in circulating hemocytes. Other transcripts and proteins identified included members of the conserved NFκB signaling pathway, putative members of the complement pathway, the carbohydrate binding protein galectin, and cephalotoxin. Quantitative PCR of complement-related genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes isolated from adult squid with colonized light organs compared to those for which the symbionts were removed. These data suggest that the presence of the symbiont influences gene expression of the cellular innate immune system of the host.

  12. Lack of association of genetic variants in genes of the endocannabinoid system with anorexia nervosa

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    Herpertz-Dahlmann Beate

    2008-11-01

    Full Text Available Abstract Background Several lines of evidence indicate that the central cannabinoid receptor 1 (CNR1 as well as the major endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH, N-acylethanolamine-hydrolyzing acid amidase (NAAA and monoglyceride lipase (MGLL are implicated in mediating the orexigenic effects of cannabinoids. The aim of this study was to analyse whether nucleotide sequence variations in the CNR1, FAAH, NAAA and MGLL genes are associated with anorexia nervosa (AN. Methods We analysed the association of a previously described (AATn repeat in the 3' flanking region of CNR1 as well as a total of 15 single nucleotide polymorphisms (SNPs representative of regions with restricted haplotype diversity in CNR1, FAAH, NAAA or MGLL in up to 91 German AN trios (patient with AN and both biological parents using the transmission-disequilibrium-test (TDT. One SNP was additionally analysed in an independent case-control study comprising 113 patients with AN and 178 normal weight controls. Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ARMS-PCR or using 3730xl capillary sequencers. Results The TDT revealed no evidence for association for any of the SNPs or the (AATn repeat with AN (all two-sided uncorrected p-values > 0.05. The lowest p-value of 0.11 was detected for the A-allele of the CNR1 SNP rs1049353 for which the transmission rate was 59% (95% confidence interval 47%...70%. Further genotyping of rs1049353 in 113 additional independent patients with AN and 178 normal weight controls could not substantiate the initial trend for association (p = 1.00. Conclusion As we found no evidence for an association of genetic variation in CNR1, FAAH, NAAA and MGLL with AN, we conclude that genetic variations in these genes do not play a major role in the etiology of AN in our study groups.

  13. PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease.

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    Elisa Ramos-Sevillano

    2016-03-01

    Full Text Available Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1 on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium--the amidase LytA--were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1-/- mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1-/- mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1-/- mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease.

  14. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    Science.gov (United States)

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-08

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  15. Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential

    Directory of Open Access Journals (Sweden)

    Siragusa Gregory R

    2011-06-01

    Full Text Available Abstract Background Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase and a holin (PF04531. Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1 strongly significant host-specific sequence variation within the endolysin, and 2 a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

  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.

    2015-09-15

    ABSTRACT

    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. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Olivier Poupel

    Full Text Available The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their

  18. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Science.gov (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  19. Enantioselective biotransformations of nitriles in organic synthesis.

    Science.gov (United States)

    Wang, Mei-Xiang

    2015-03-17

    The hydration and hydrolysis of nitriles are valuable synthetic methods used to prepare carboxamides and carboxylic acids. However, chemical hydration and hydrolysis of nitriles involve harsh reaction conditions, have low selectivity, and generate large amounts of waste. Therefore, researchers have confined the scope of these reactions to simple nitrile substrates. However, biological transformations of nitriles are highly efficient, chemoselective, and environmentally benign, which has led synthetic organic chemists and biotechologists to study these reactions in detail over the last two decades. In nature, biological systems degrade nitriles via two distinct pathways: nitrilases catalyze the direct hydrolysis of nitriles to afford carboxylic acids with release of ammonia, and nitrile hydratases catalyze the conversion of nitriles into carboxamides, which then furnish carboxylic acids via hydrolysis in the presence of amidases. Researchers have subsequently developed biocatalytic methods into useful industrial processes for the manufacture of commodity chemicals, including acrylamide. Since the late 1990s, research by my group and others has led to enormous progress in the understanding and application of enantioselective biotransformations of nitriles in organic synthesis. In this Account, I summarize the important advances in enantioselective biotransformations of nitriles and amides, with a primary focus on research from my laboratory. I describe microbial whole-cell-catalyzed kinetic resolution of various functionalized nitriles, amino- and hydroxynitriles, and nitriles that contain small rings and the desymmetrization of prochiral and meso dinitriles and diamides. I also demonstrate how we can apply the biocatalytic protocol to synthesize natural products and bioactive compounds. These nitrile biotransformations offer an attractive and unique protocol for the enantioselective synthesis of polyfunctionalized organic compounds that are not readily obtainable by

  20. Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253.

    Science.gov (United States)

    Tucker, Trudy-Ann; Crow, Sidney A; Pierce, George E

    2012-11-01

    Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202 units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55 °C (45 and 23 %, respectively) than cells grown in the presence of glucose or fructose (19 and 10 %, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55 °C, as cells grown in the presence of glucose retained 40 % NHase activity as opposed to 19 % without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and

  1. A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

    Science.gov (United States)

    Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

    2013-07-12

    Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

  2. Bifidobacterium breve C50 secretes lipoprotein with CHAP domain recognized in aggregated form by TLR2.

    Science.gov (United States)

    Scuotto, Angelo; Djorie, Serge; Colavizza, Michel; Romond, Pierre-Charles; Romond, Marie-Bénédicte

    2014-12-01

    Extracellular components secreted by Bifidobacterium breve C50 can induce maturation, high IL-10 production and prolonged survival of dendritic cells via a TLR2 pathway. In this study, the components were isolated from the supernatant by gel filtration chromatography. Antibodies raised against the major compounds with molecular weight above 600 kDa (Bb C50BC) also recognized compounds of lower molecular weight (200–600 kDa). TLR2 and TLR6 bound to the components already recognized by the antibodies. Trypsin digestion of Bb C50BC released three major peptides whose sequences displayed close similarities to a putative secreted protein with a CHAP amidase domain from B. breve. The 1300-bp genomic region corresponding to the hypothetical protein was amplified by PCR. The deduced polypeptide started with an N-terminal signal sequence of 45 amino acids, containing the lipobox motif (LAAC) with the cysteine in position 25, and 2 positively charged residues within the first 14 residues of the signal sequence. Lipid detection in Bb C50BC by GC/MS further supported the implication of a lipoprotein. Sugars were also detected in Bb C50BC. Close similarity with the glucan-binding protein B from Bifidobacterium animalis of two released peptides from Bb C50BC protein suggested that glucose moieties, possibly in glucan form, could be bound to the lipoprotein. Finally, heating at 100 °C for 5 min led to the breakdown of Bb C50BC in compounds of molecular weight below 67 kDa, which suggested that Bb C50BC was an aggregate. One might assume that a basic unit was formed by the lipoprotein bound putatively to glucan. Besides the other sugars and hexosamines recognized by galectin 1 were localized at the surface of the Bb C50BC aggregate. In conclusion, the extracellular components secreted by B. breve C50 were constituted of a lipoprotein putatively associated with glucose moieties and acting in an aggregating form as an agonist of TLR2/TLR6.

  3. Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

    Science.gov (United States)

    Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

    2016-02-02

    surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Characterization and complete genome sequence analysis of a novel virulent Siphoviridae phage against Staphylococcus aureus isolated from bovine mastitis in Xinjiang, China.

    Science.gov (United States)

    Zhang, Qian; Xing, Shaozhen; Sun, Qiang; Pei, Guangqian; Cheng, Shi; Liu, Yannan; An, Xiaoping; Zhang, Xianglilan; Qu, Yonggang; Tong, Yigang

    2017-06-01

    creation of a new lineage, thus adding to the knowledge on the diversity of Staphylococcus phages. An N-acetylmuramoyl-L-alanine amidase gene and several conserved genes were predicted, while no virulence or antibiotic resistance genes were identified. This study isolated and characterized a novel S. aureus phage vB_SauS_IMEP5, and our findings suggest that this phage may be potentially utilized as a therapeutic or prophylactic candidate against S.aureus infections.

  5. METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

    Energy Technology Data Exchange (ETDEWEB)

    John J. Kilbane II

    2004-10-01

    The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project was focused on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate deaminase. The objective of the final phase of the project will be to develop derivative C-N bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments resulted in the isolation of microbial cultures that utilize aromatic amides as sole nitrogen sources, several amidase genes were cloned and were included in directed evolution experiments to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. During the second year of the project (October, 2003-September, 2004) enrichment culture experiments succeeded in isolating a mixed bacterial culture that can utilize 2-aminobiphenyl as a sole nitrogen source, directed evolution experiments were focused on the aniline dioxygenase enzyme that is capable of deaminating aniline, and expression vectors were constructed to enable the expression of genes encoding C-N bond cleaving enzymes in Rhodococcus hosts. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the

  6. Dual-lifetime referencing (DLR: a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

    Directory of Open Access Journals (Sweden)

    Boniello Caterina

    2012-03-01

    Full Text Available Abstract Background Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier. Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation. Results The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II tris(4,7-diphenyl-1,10-phenantroline, abbreviated Ru(dpp, as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance. The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under

  7. The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

    Science.gov (United States)

    Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

    2017-06-01

    Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds