Organophosphorus poisoning (acute).

PubMed

Blain, Peter G

2011-05-17

Acetylcholinesterase inhibition by organophosphorus pesticides or organophosphate nerve agents can cause acute parasympathetic system dysfunction, muscle weakness, seizures, coma, and respiratory failure. Prognosis depends on the dose and relative toxicity of the specific compound, as well as pharmacokinetic factors. We conducted a systematic review and aimed to answer the following clinical question: What are the effects of treatments for acute organophosphorus poisoning? We searched: Medline, Embase, The Cochrane Library, and other important databases up to April 2010 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). We found 62 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. In this systematic review we present information relating to the effectiveness and safety of the following interventions: activated charcoal (single or multiple doses), alpha(2) adrenergic receptor agonists, atropine, benzodiazepines, butyrylcholinesterase replacement therapy, cathartics, extracorporeal clearance, gastric lavage, glycopyrronium bromide (glycopyrrolate), ipecacuanha (ipecac), magnesium sulphate, milk or other home remedy immediately after ingestion, N-methyl-D-aspartate receptor antagonists, organophosphorus hydrolases, oximes, removing contaminated clothes and washing the poisoned person, and sodium bicarbonate.

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

NASA Astrophysics Data System (ADS)

Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

2014-07-01

We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

PubMed Central

Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

2015-01-01

Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

PubMed

Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

2015-07-28

Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

PubMed Central

Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

2015-01-01

ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

New insights on molecular interactions of organophosphorus pesticides with esterases.

PubMed

Mangas, Iris; Estevez, Jorge; Vilanova, Eugenio; França, Tanos Celmar Costa

2017-02-01

Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the

Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by Pichia pastoris without Methanol Induction.

PubMed

Bai, Yun-Peng; Luo, Xiao-Jing; Zhao, Yu-Lian; Li, Chun-Xiu; Xu, Dian-Sheng; Xu, Jian-He

2017-10-18

The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPH M9 , an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 g protein /L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L -1 h -1 . PoOPH M9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 μmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPH M9 in tap water after 35 min washing. The efficient production of the highly active PoOPH M9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

Proteomic analysis of adducted butyrylcholinesterase for biomonitoring organophosphorus exposures

PubMed Central

Marsillach, Judit; Hsieh, Edward J.; Richter, Rebecca J.; MacCoss, Michael J.; Furlong, Clement E.

2014-01-01

Organophosphorus (OP) compounds include a broad group of toxic chemicals such as insecticides, chemical warfare agents and antiwear agents. The liver cytochromes P450 bioactivate many OPs to potent inhibitors of serine hydrolases. Cholinesterases were the first OP targets discovered and are the most studied. They are used to monitor human exposures to OP compounds. However, the assay that is currently used has limitations. The mechanism of action of OP compounds is the inhibition of serine hydrolases by covalently modifying their active-site serine. After structural rearrangement, the complex OP inhibitor-enzyme is irreversible and will remain in circulation until the modified enzyme is degraded. Mass spectrometry is a sensitive technology for analyzing protein modifications, such as OP-adducted enzymes. These analyses also provide some information about the nature of the OP adduct. Our aim is to develop high-throughput protocols for monitoring OP exposures using mass spectrometry. PMID:23123252

Actin polymerization drives septation of Listeria monocytogenes namA hydrolase mutants, demonstrating host correction of a bacterial defect.

PubMed

Alonzo, Francis; McMullen, P David; Freitag, Nancy E

2011-04-01

The Gram-positive bacterial cell wall presents a structural barrier that requires modification for protein secretion and large-molecule transport as well as for bacterial growth and cell division. The Gram-positive bacterium Listeria monocytogenes adjusts cell wall architecture to promote its survival in diverse environments that include soil and the cytosol of mammalian cells. Here we provide evidence for the enzymatic flexibility of the murein hydrolase NamA and demonstrate that bacterial septation defects associated with a loss of NamA are functionally complemented by physical forces associated with actin polymerization within the host cell cytosol. L. monocytogenes ΔnamA mutants formed long bacterial chains during exponential growth in broth culture; however, normal septation could be restored if mutant cells were cocultured with wild-type L. monocytogenes bacteria or by the addition of exogenous NamA. Surprisingly, ΔnamA mutants were not significantly attenuated for virulence in mice despite the pronounced exponential growth septation defect. The physical force of L. monocytogenes-mediated actin polymerization within the cytosol was sufficient to sever ΔnamA mutant intracellular chains and thereby enable the process of bacterial cell-to-cell spread so critical for L. monocytogenes virulence. The inhibition of actin polymerization by cytochalasin D resulted in extended intracellular bacterial chains for which septation was restored following drug removal. Thus, despite the requirement for NamA for the normal septation of exponentially growing L. monocytogenes cells, the hydrolase is essentially dispensable once L. monocytogenes gains access to the host cell cytosol. This phenomenon represents a notable example of eukaryotic host cell complementation of a bacterial defect.

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

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. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm 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 structuremore » 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.Peptidoglycan 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 analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

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. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm 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 structuremore » 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.Peptidoglycan 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 analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

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-A 2pm (A 2pm 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 consistingmore » 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 analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling

Catalytic degradation of organophosphorous nerve agent simulants by polymer beads@graphene oxide with organophosphorus hydrolase-like activity based on rational design of functional bimetallic nuclear ligand.

PubMed

Ma, Xuejuan; Zhang, Lin; Xia, Mengfan; Zhang, Xiaohong; Zhang, Yaodong

2018-05-15

The degradation of organophosphorous nerve agents is of primary concern due to the severe toxicity of these agents. Based on the active center of organophosphorus hydrolase (OPH), a bimetallic nuclear ligand, (5-vinyl-1,3-phenylene)bis(di(1H-imidazol-2-yl) methanol) (VPIM), was designed and synthesized, which contains four imidazole groups to mimic the four histidines at OPH active center. By grafting VPIM on graphene oxide (GO) surface via polymerization, the VPIM-polymer beads@GO was produced. The obtained OPH mimics has an impressive activity in dephosphorylation reactions (turnover frequency (TOF) towards paraoxon: 2.3 s -1 ). The synergistic catalytic effect of the bimetallic Zn 2+ nuclear center and carboxyl groups on surface of GO possibly contributes to the high hydrolysis on organophosphate substrate. Thus, a biomimetic catalyst for efficient degradation of some organophosphorous nerve agent simulants, such as paraoxon and chlorpyrifos, was prepared by constructing catalytic active sites. The proposed mechanism and general synthetic strategy open new avenues for the engineering of functional GOs for biomimetic catalysts. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular Dynamics Simulations of Acylpeptide Hydrolase Bound to Chlorpyrifosmethyl Oxon and Dichlorvos

PubMed Central

Jin, Hanyong; Zhou, Zhenhuan; Wang, Dongmei; Guan, Shanshan; Han, Weiwei

2015-01-01

Acylpeptide hydrolases (APHs) catalyze the removal of N-acylated amino acids from blocked peptides. Like other prolyloligopeptidase (POP) family members, APHs are believed to be important targets for drug design. To date, the binding pose of organophosphorus (OP) compounds of APH, as well as the different OP compounds binding and inducing conformational changes in two domains, namely, α/β hydrolase and β-propeller, remain poorly understood. We report a computational study of APH bound to chlorpyrifosmethyl oxon and dichlorvos. In our docking study, Val471 and Gly368 are important residues for chlorpyrifosmethyl oxon and dichlorvos binding. Molecular dynamics simulations were also performed to explore the conformational changes between the chlorpyrifosmethyl oxon and dichlorvos bound to APH, which indicated that the structural feature of chlorpyrifosmethyl oxon binding in APH permitted partial opening of the β-propeller fold and allowed the chlorpyrifosmethyl oxon to easily enter the catalytic site. These results may facilitate the design of APH-targeting drugs with improved efficacy. PMID:25794283

Relieving Mipafox Inhibition in Organophosphorus Acid Anhydrolase by Rational Design

DTIC Science & Technology

2013-03-01

acid anhydrolase (OPAA, EC 3.1.8.2) was purified from halophilic Alteromonas sp. bacteria. OPPA displayed hydrolysis activity against several highly...2010, 49, 547–559. 3. DeFrank, J.J.; Cheng, T.-C. Purification and Properties of Organophosphorus Acid Anhydrolase from a Halophilic Bacterial

Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...

Enzymatic Decontamination of Environmental Organophosphorus Compounds

DTIC Science & Technology

2006-12-04

ABSTRACT (Maximum 200 words) The abstract is below since many authors do not follow the 200 word limit 14. SUBJECT TERMS organophosphorus compounds ...5404 Enzymatic decontamination of environmental organophosphorus compounds REPORT DOCUMENTATION PAGE 18. SECURITY CLASSIFICATION ON THIS PAGE...239-18 298-102 15. NUMBER OF PAGES 20. LIMITATION OF ABSTRACT UL - 4-Dec-2006 Enzymatic decontamination of environmental organophosphorus compounds

Exploration of the chlorpyrifos escape pathway from acylpeptide hydrolases using steered molecular dynamics simulations.

PubMed

Wang, Dongmei; Jin, Hanyong; Wang, Junling; Guan, Shanshan; Zhang, Zuoming; Han, Weiwei

2016-01-01

Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer's disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family.

A proton wire and water channel revealed in the crystal structure of isatin hydrolase.

PubMed

Bjerregaard-Andersen, Kaare; Sommer, Theis; Jensen, Jan K; Jochimsen, Bjarne; Etzerodt, Michael; Morth, J Preben

2014-08-01

The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycoside hydrolases having multiple hydrolase activities

DOEpatents

Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

2017-08-08

Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

Zebrafish Models for Human Acute Organophosphorus Poisoning.

PubMed

Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

2015-10-22

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases

PubMed Central

McGregor, Nicholas; Yin, Victor; Tung, Ching-Chieh; Van Petegem, Filip; Brumer, Harry

2016-01-01

SUMMARY The xyloglucan endo-transglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodelling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endo-glycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endo-glucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endo-tranglycosylase (XET), and a xyloglucan endo-hydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species. PMID:27859885

Neurotoxic disorders of organophosphorus compounds and their managements.

PubMed

Balali-Mood, Mahdi; Balali-Mood, Kia

2008-01-01

Organophosphorus compounds have been used as pesticides and as chemical warfare nerve agents. The mechanism of toxicity of organophosphorus compounds is the inhibition of acetylcholinesterase, which results in accumulation of acetylcholine and the continued stimulation of acetylcholine receptors. Therefore, they are also called anticholinesterase agents. Organophosphorus pesticides have largely been used worldwide, and poisoning by these agents, particularly in developing countries, is a serious health problem. Organophosphorus nerve agents were used by Iraqi army against Iranian combatants and even civilian population in 1983 - 1988. They were also used for chemical terrorism in Japan in 1994 - 1995. Their use is still a constant threat to the population. Therefore, medical and health professionals should be aware and learn more about the toxicology and proper management of organophosphorus poisoning. Determination of acetylcholinesterase and butyrylcholinesterase activity in blood remains a mainstay for the fast initial screening of organophosphorus compounds but lacks sensitivity and specificity. Quantitative analysis of organophosphorus compounds and their degradation products in plasma and urine by mass spectrometric methods may prove exposure but is expensive and is limited to specialized laboratories. However, history of exposure to organophosphorous compounds and clinical manifestations of a cholinergic syndrome are sufficient for management of the affected patients. The standard management of poisoning with organophosphorous compounds consists of decontamination, and injection of atropine sulfate with an oxime. Recent advances on treatment of organophosphorus pesticides poisoning revealed that blood alkalinization with sodium bicarbonate and also magnesium sulfate as adjunctive therapies are promising. Patients who receive prompt proper treatment usually recover from acute toxicity but may suffer from neurologic complications.

Multidimensional profiling platforms reveal metabolic dysregulation caused by organophosphorus pesticides.

PubMed

Medina-Cleghorn, Daniel; Heslin, Ann; Morris, Patrick J; Mulvihill, Melinda M; Nomura, Daniel K

2014-02-21

We are environmentally exposed to countless synthetic chemicals on a daily basis, with an increasing number of these chemical exposures linked to adverse health effects. However, our understanding of the (patho)physiological effects of these chemicals remains poorly understood, due in part to a general lack of effort to systematically and comprehensively identify the direct interactions of environmental chemicals with biological macromolecules in mammalian systems in vivo. Here, we have used functional chemoproteomic and metabolomic platforms to broadly identify direct enzyme targets that are inhibited by widely used organophosphorus (OP) pesticides in vivo in mice and to determine metabolic alterations that are caused by these chemicals. We find that these pesticides directly inhibit over 20 serine hydrolases in vivo leading to widespread disruptions in lipid metabolism. Through identifying direct biological targets of OP pesticides, we show heretofore unrecognized modes of toxicity that may be associated with these agents and underscore the utility of using multidimensional profiling approaches to obtain a more complete understanding of toxicities associated with environmental chemicals.

Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.

PubMed

Xu, Qingping; Chiu, Hsiu-Ju; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Lesley, Scott A; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

2014-01-09

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state). © 2013.

Organophosphorus and carbamate pesticides

USGS Publications Warehouse

Glaser, L.C.

1999-01-01

The insecticidal properties of organophosphorus (OP) and carbamate compounds were first discovered in the 1930s, and the compounds were developed for pesticide use in the 1940s. They have been used increasingly since the 1970s when environmentally persistent organochlorine pesticides, such as DDT and dieldrin, were banned for use in the United States. Organophosphorus and carbamate pesticides are generally short-lived in the environment (usually lasting only days to months instead of years) and, generally, chemical breakdown is accelerated as temperatures or pH or both increase.

Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Wataru; Ochiai, Akihito; Momma, Keiko

Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 deg. C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 A resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, {alpha} and {beta}. Amore » deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase.« less

The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae.

PubMed

Jacq, Maxime; Arthaud, Christopher; Manuse, Sylvie; Mercy, Chryslène; Bellard, Laure; Peters, Katharina; Gallet, Benoit; Galindo, Jennifer; Doan, Thierry; Vollmer, Waldemar; Brun, Yves V; VanNieuwenhze, Michael S; Di Guilmi, Anne Marie; Vernet, Thierry; Grangeasse, Christophe; Morlot, Cecile

2018-05-15

Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.

Purification and Characterization of a Novel Chlorpyrifos Hydrolase from Cladosporium cladosporioides Hu-01

PubMed Central

Chen, Shaohua; Hu, Meiying; Luo, Jianjun; Li, Yanan

2012-01-01

Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2+, Fe3+, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5–10% inhibition) were observed in the presence of Mn2+, Zn2+, Cu2+, Mg2+, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min−1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus. PMID:22693630

[Meta-analysis of association between organophosphorus pesticides and aplastic anemia].

PubMed

Zhang, Ji; Yang, Tubao

2015-09-01

To evaluate the association between organophosphorus pesticides and aplastic anemia, and provide scientific evidence for the primary prevention of aplastic anemia. The published papers of case control studies on the association between organophosphorus pesticides and aplastic anemia from January 1990 to August 2014 were collected from Chinese BioMedical Literature Base (CBM), Chinese National Knowledge Infrastructure (CNKI), PubMed and EMBASE. The papers which met the inclusion criteria were evaluated. The pooled odds ratios (OR) and 95% confidence interval (CI) of organophosphorus pesticides were calculated with software Review Manager 5.0. Subgroup analysis were conducted for different population and different usage of organophosphorus pesticides. A total of 9 papers were selected, involving 5 833 subjects (1 404 cases and 4 429 controls). The results showed that organophosphorus pesticides could increase the risk of aplastic anemia (OR=1.97, 95% CI: 1.60-2.44) . Subgroup analysis showed that Asian (OR=2.01, 95% CI: 1.52-2.66) had higher risk of aplastic anemia than American or European (OR=1.93, 95% CI: 1.39-2.67) . Using pure organophosphorus pesticides (OR=2.15, 95% CI: 1.60-2.88) was more prone to cause aplastic anemia than using the mixture of organophosphorus pesticides (OR=1.82, 95% CI: 1.34-2.47). The analysis indicated that organophosphorus pesticides might be a risk factor for aplastic anemia. Reducing organophosphorus pesticides exposure in daily life and industrial or agricultural production could prevent the incidence of aplastic anemia.

The Vital Function of Fe3O4@Au nanocomposites for Hydrolase Biosensor Design and Its Application in Detection of Methyl Parathion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Yuting; Zhang, Weiying; Lin, Yuehe

A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core was synthesized using cysteamine (SH–NH2) as linker, and characterized by TEM, XPS, UV and electrochemistry. Then a hydrolase biosensor, based on self-assembly of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for sensitive and selective detection of the organophosphorus pesticide (OP) methyl parathion. The magnetic nanocomposite provides an easy way to construct the enzyme biosensor by simply exerting an external magnetic field, and also provides a simple way to renew the electrode surface by removing the magnet. Unlike inhibition-based enzyme biosensors, the hydrolase is not poisoned bymore » OPs and thus is reusable for continuous measurement. AuNPs not only provide a large surface area, high loading efficiency and fast electron transfer, but also stabilize the enzyme through electrostatic interactions. The MPH biosensor shows rapid response and high selectivity for detection of methyl parathion, with a linear range from 0.5 to 1000 ng/mL and a detection limit of 0.1 ng/mL. It also shows acceptable reproducibility and stability. The simplicity and ease of operation of the proposed method has great potential for on-site detection of P–S containing pesticides and provides a promising strategy to construct a robust biosensor.« less

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

PubMed Central

Baker, Perrin; Hill, Preston J.; Snarr, Brendan D.; Alnabelseya, Noor; Pestrak, Matthew J.; Lee, Mark J.; Jennings, Laura K.; Tam, John; Melnyk, Roman A.; Parsek, Matthew R.; Sheppard, Donald C.; Wozniak, Daniel J.; Howell, P. Lynne

2016-01-01

Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics. PMID:27386527

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.

PubMed

Baker, Perrin; Hill, Preston J; Snarr, Brendan D; Alnabelseya, Noor; Pestrak, Matthew J; Lee, Mark J; Jennings, Laura K; Tam, John; Melnyk, Roman A; Parsek, Matthew R; Sheppard, Donald C; Wozniak, Daniel J; Howell, P Lynne

2016-05-01

Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

Peptidoglycan Hydrolases of Escherichia coli

PubMed Central

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

Chlamydia trachomatis CT771 (nudH) is an asymmetric Ap4A hydrolase.

PubMed

Barta, Michael L; Lovell, Scott; Sinclair, Amy N; Battaile, Kevin P; Hefty, P Scott

2014-01-14

Asymmetric diadenosine 5',5‴-P(1),P(4)-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologues to CT771 within environmental Chlamydiales that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 Å and 1.9 Å resolution, respectively. The structure of CT771 shows a αβα-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap4A hydrolase from Caenorhabditis elegans. These structures represent only the second Ap4A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap4A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap4A hydrolase (nudH).

Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in glycoside hydrolase family 1.

PubMed Central

Marques, Ana Rita; Coutinho, Pedro M; Videira, Paula; Fialho, Arsénio M; Sá-Correia, Isabel

2003-01-01

The Sphingomonas paucimobilis beta-glucosidase Bgl1 is encoded by the bgl1 gene, associated with an 1308 bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bgl1 protein through the inner membrane to the periplasmic space. A His(6)-tagged 44.3 kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bgl1 gene lacking the region encoding the putative signal peptide. Mature beta-glucosidase Bgl1 is specific for aryl-beta-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bgl1 and other members of the glycoside hydrolase (GH) family 1 enzymes. At subsite -1, the conserved residues required for catalysis by GH1 enzymes are present in Bgl1 with only minor differences. Major differences are found at subsite +1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH1 enzymes, revealing an absence of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bgl1, the first characterized beta-glucosidase from the genus Sphingomonas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin. PMID:12444924

Organophosphorus Insecticide Pharmacokinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timchalk, Charles

2010-01-01

This chapter highlights a number of current and future applications of pharmacokinetics to assess organophosphate (OP) insecticide dosimetry, biological response and risk in humans exposed to these agents. Organophosphates represent a large family of pesticides where insecticidal as well as toxicological mode of action is associated with their ability to target and inhibit acetylcholinesterase (AChE). Pharmacokinetics entails the quantitative integration of physiological and metabolic processes associated with the absorption, distribution, metabolism and excretion (ADME) of drugs and xenobiotics. Pharmacokinetic studies provide important data on the amount of toxicant delivered to a target site as well as species-, age-, gender-specific andmore » dose-dependent differences in biological response. These studies have been conducted with organophosphorus insecticides in multiple species, at various dose levels, and across different routes of exposure to understand their in vivo pharmacokinetics and how they contribute to the observed toxicological response. To access human exposure to organophosphorus insecticides, human pharmacokinetic studies have been conducted and used to develop biological monitoring strategies based on the quantitation of key metabolites in biological fluids. Pharmacokinetic studies with these insecticides are also useful to facilitate extrapolation of dosimetry and biological response from animals to humans and for the assessment of human health risk. In this regard, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models are being utilized to assess risk and understand the toxicological implications of known or suspected exposures to various insecticides. In this chapter a number of examples are presented that illustrate the utility and limitation of pharmacokinetic studies to address human health concerns associated with organophosphorus insecticides.« less

Unraveling the stereochemical and dynamic aspects of the catalytic site of bacterial peptidyl-tRNA hydrolase

PubMed Central

Kabra, Ashish; Shahid, Salman; Pal, Ravi Kant; Yadav, Rahul; Pulavarti, S.V.S. Rama Krishna; Jain, Anupam; Tripathi, Sarita; Arora, Ashish

2017-01-01

Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies of Vibrio cholerae Pth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme. PMID:28096445

EFFECT OF ORGANOPHOSPHORUS FLAME RETARDANTS ON NEURONAL DEVELOPMENT IN VITRO

EPA Science Inventory

The increased use of organophosphorus compounds as alternatives to brominated flame retardants (BFRs) has led to widespread human exposure, There is, however, limited information on their potential health effects. This study compared the effects of nii ne organophosphorus flame...

γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

PubMed

Mamberti, Stefania; Prati, Paola; Cremaschi, Paolo; Seppi, Claudio; Morelli, Carlo F; Galizzi, Alessandro; Fabbi, Massimo; Calvio, Cinzia

2015-01-01

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Active site and laminarin binding in glycoside hydrolase family 55

DOE PAGES

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

2015-03-09

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

Management of acute organophosphorus pesticide poisoning.

PubMed

Eddleston, Michael; Buckley, Nick A; Eyer, Peter; Dawson, Andrew H

2008-02-16

Organophosphorus pesticide self-poisoning is an important clinical problem in rural regions of the developing world, and kills an estimated 200,000 people every year. Unintentional poisoning kills far fewer people but is a problem in places where highly toxic organophosphorus pesticides are available. Medical management is difficult, with case fatality generally more than 15%. We describe the limited evidence that can guide therapy and the factors that should be considered when designing further clinical studies. 50 years after first use, we still do not know how the core treatments--atropine, oximes, and diazepam--should best be given. Important constraints in the collection of useful data have included the late recognition of great variability in activity and action of the individual pesticides, and the care needed cholinesterase assays for results to be comparable between studies. However, consensus suggests that early resuscitation with atropine, oxygen, respiratory support, and fluids is needed to improve oxygen delivery to tissues. The role of oximes is not completely clear; they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning. Small studies suggest benefit from new treatments such as magnesium sulphate, but much larger trials are needed. Gastric lavage could have a role but should only be undertaken once the patient is stable. Randomised controlled trials are underway in rural Asia to assess the effectiveness of these therapies. However, some organophosphorus pesticides might prove very difficult to treat with current therapies, such that bans on particular pesticides could be the only method to substantially reduce the case fatality after poisoning. Improved medical management of organophosphorus poisoning should result in a reduction in worldwide deaths from suicide.

Management of acute organophosphorus pesticide poisoning

PubMed Central

Eddleston, Michael; Buckley, Nick A; Eyer, Peter; Dawson, Andrew H

2008-01-01

Summary Organophosphorus pesticide self-poisoning is an important clinical problem in rural regions of the developing world, and kills an estimated 200 000 people every year. Unintentional poisoning kills far fewer people but is a problem in places where highly toxic organophosphorus pesticides are available. Medical management is difficult, with case fatality generally more than 15%. We describe the limited evidence that can guide therapy and the factors that should be considered when designing further clinical studies. 50 years after first use, we still do not know how the core treatments—atropine, oximes, and diazepam—should best be given. Important constraints in the collection of useful data have included the late recognition of great variability in activity and action of the individual pesticides, and the care needed cholinesterase assays for results to be comparable between studies. However, consensus suggests that early resuscitation with atropine, oxygen, respiratory support, and fluids is needed to improve oxygen delivery to tissues. The role of oximes is not completely clear; they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning. Small studies suggest benefit from new treatments such as magnesium sulphate, but much larger trials are needed. Gastric lavage could have a role but should only be undertaken once the patient is stable. Randomised controlled trials are underway in rural Asia to assess the effectiveness of these therapies. However, some organophosphorus pesticides might prove very difficult to treat with current therapies, such that bans on particular pesticides could be the only method to substantially reduce the case fatality after poisoning. Improved medical management of organophosphorus poisoning should result in a reduction in worldwide deaths from suicide. PMID:17706760

Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

PubMed Central

Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

2011-01-01

Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

PubMed

Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

2014-01-01

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

[Perspectives in the treatments of poisonings by organophosphorus insecticides and warfare nerve agents].

PubMed

Sogorb-Sánchez, M A; Vilanova-Gisbert, E; Carrera-González, V

Organophosphorus compounds are worldwide employed as insecticides and are yearly responsible of several millions of poisonings. The chemical structure of most of the warfare nerve agents also corresponds with an organophosphorus compound. Organophosphorus insecticides and warfare nerve agents exert their main toxicological effects through inhibition of acetylcholinesterase. Current treatments of patients poisoned with organophosphorus compounds include atropine (in order to protect muscarinic receptors), oximes (in order to accelerate the reactivation of the inhibited acetylcholinesterase) and benzodiazepines (in order to avoid convulsions). The administration of phosphotriesterases (enzymes involved in the detoxication of organophosphorus compounds through hydrolysis) is a very effective treatment against poisonings by organophosphorus insecticides and warfare nerve agents. There are experimental preventive treatments based on the simultaneous administration of carbamates and certain antimuscarinic drugs, different from atropine, which notably improve the efficacy of the classical treatments applied after poisonings by warfare nerve agents. The treatments based in the administration of phosphotriesterases might be the response to the call of the World Health Organization for searching new treatments with capability to reduce the high mortality recorded in the cases of poisonings by organophosphorus compounds. These treatments can be applied in a preventive way without the intrinsic neurotoxicity associated to the preventive treatments based on carbamates and antimuscarinic drugs. Therefore, these treatments are specially interesting for people susceptible to suffer severe exposures, i.e. sprayers in the farms.

Epoxide hydrolases: structure, function, mechanism, and assay.

PubMed

Arand, Michael; Cronin, Annette; Adamska, Magdalena; Oesch, Franz

2005-01-01

Epoxide hydrolases are a class of enzymes important in the detoxification of genotoxic compounds, as well as in the control of physiological signaling molecules. This chapter gives an overview on the function, structure, and enzymatic mechanism of structurally characterized epoxide hydrolases and describes selected assays for the quantification of epoxide hydrolase activity.

PROTEIN ADDUCTS AS BIOMAKERS OF EXPOSURE TO ORGANOPHOSPHORUS COMPOUNDS

PubMed Central

Marsillach, Judit; Costa, Lucio G.; Furlong, Clement E.

2013-01-01

Exposure to organophosphorus (OP) compounds can lead to serious neurological damage or death. Following bioactivation by the liver cytochromes P450, the OP metabolites produced are potent inhibitors of serine active-site enzymes including esterases, proteases and lipases. OPs may form adducts on other cellular proteins. Blood cholinesterases (ChEs) have long served as biomarkers of OP exposure in humans. However, the enzymatic assays used for biomonitoring OP exposures have several drawbacks. A more useful approach will focus on multiple biomarkers and avoid problems with the enzymatic activity assays. OP inhibitory effects result from a covalent bond with the active-site serine of the target enzymes. The serine OP adducts become irreversible following a process referred to as aging where one alkyl group dissociates over variable lengths of time depending on the OP adduct. The OP-adducted enzyme then remains in circulation until it is degraded, allowing for a longer window of detection compared with direct analysis of OPs or their metabolites. Mass spectrometry (MS) provides a very sensitive method for identification of post-translational protein modifications. MS analyses of the percentage adduction of the active-site serine of biomarker proteins such as ChEs will eliminate the need for basal activity levels of the individual and will provide for a more accurate determination of OP exposure. MS analysis of biomarker proteins also provides information about the OP that has caused inhibition. Other useful biomarker proteins include other serine hydrolases, albumin, tubulin and transferrin. PMID:23261756

Novel organophosphorus scaffolds of urease inhibitors obtained by substitution of Morita-Baylis-Hillman adducts with phosphorus nucleophiles.

PubMed

Ntatsopoulos, Vassilis; Vassiliou, Stamatia; Macegoniuk, Katarzyna; Berlicki, Łukasz; Mucha, Artur

2017-06-16

The reactivity of Morita-Baylis-Hillman allyl acetates was employed to introduce phosphorus-containing functionalities to the side chain of the cinnamic acid conjugated system by nucleophilic displacement. The proximity of two acidic groups, the carboxylate and phosphonate/phosphinate groups, was necessary to form interactions in the active site of urease by recently described inhibitor frameworks. Several organophosphorus scaffolds were obtained and screened for inhibition of the bacterial urease, an enzyme that is essential for survival of urinary and gastrointestinal tract pathogens. α-Substituted phosphonomethyl- and 2-phosphonoethyl-cinnamate appeared to be the most potent and were further optimized. As a result, one of the most potent organophosphorus inhibitors of urease, α-phosphonomethyl-p-methylcinnamic acid, was identified, with K i  = 0.6 μM for Sporosarcina pasteurii urease. High complementarity to the enzyme active site was achieved with this structure, as any further modifications significantly decreased its affinity. Finally, this work describes the challenges faced in developing ligands for urease. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Levels of organophosphorus pesticides in medicinal plants commonly consumed in Iran

PubMed Central

2012-01-01

The frequent occurrence of pesticide residues in herbal materials was indicated by previous studies. In this study, the concentration of some of the organophosphorus pesticides including parathion, malathion, diazinon and pirimiphos methyl in different kinds of medicinal plants were determined. The samples were collected randomly from ten local markets of different areas of Iran. At the detection limit of 0.5 ng g-1, parathion and pirimiphos methyl were not detected in any of the samples. Some amounts of malathion and diazinon were found in Zataria, Matricaria chamomile, Spearmint and Cumin Seed samples while, the concentrations of target organophosphorus pesticides in Borage samples were below the detection limits of the methods which could be a result of intensive transformation of organophosphorus pesticides by Borage. In addition the organophosphorus pesticides were detected in all of the samples below the maximum residue levels (MRLs) proposed by the international organizations. PMID:23351610

QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis

NASA Technical Reports Server (NTRS)

Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

1985-01-01

Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of catonic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrogen hydrolysis rate constant.

Partial purification and characterization of an inducible indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, Jyh-Ching; Cohen, J.D.; Mulbry, W.W.

1996-11-01

Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH{sub 4}){sub 2}SO{sub 4}, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusivelymore » high substrate specificity for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg{sup 2+} the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO{sub 4}; the activity was increased by 40% with 1mM MnSO{sub 4}. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 45 refs., 5 figs., 3 tabs.« less

Biodegradation of organophosphorus pesticides by soil bacteria

NASA Astrophysics Data System (ADS)

de Pasquale, C.; Fodale, R.; Lo Piccolo, L.; Palazzolo, E.; Alonzo, G.; Quatrini, P.

2009-04-01

A number of studies in the 1980s and 1990s showed that crop-protection products, applied to drained fields, could move downwards through the soil profile and to the groundwater. Organophosphorus insecticides (OPs) are used all over the world for crop protection, for other agricultural practices such as sheep dipping and, in aquaculture, for the control of sea lice. Ops besides showing a specific neurotoxicity and have also been related to various modern diseases, including Creutzfeldt-Jakob (CJD) and the Gulf War syndrome. Although OPs are less persistent than Organoclorine pesticides (OCs), they still constitute an environmental risks thus increasing the social concern about their levels in soils, surface waters, and ground waters. Degradation of OPs by microorganisms has been assessed for a few bacterial strains. In the present study the OPs degrading potential of indigenous soil microorganisms was investigated. Using enrichment cultures in which parathion was the only C and energy sources many bacterial strains were isolated from OPs contaminated and pristine agricultural soils characterized by different physico-chemical properties. More than 40 potential OPs degraders were isolated and grouped in operational taxonomic units (OTU) using analysis of polymorphism showed by the ribosomal internal transcribed spacer (ITS). Partial sequencing of 16S rRNA gene of representative isolates of each OTU revealed that most of them belong to Proteobacteria and Actinobacteria. All the analyzed soils showed the presence of putative OPs degraders: the highest diversity was found in organic cultivated soils, the lowest in chemically cultivated soils. Degradation of different OPs, characterized by different physical and chemical properties, was obtained by different selected representative strains using SPME GC-MS analysis on water and soil microcosms. The results showed that, after the incubation period, the amount of pesticide residues were in the range 20-80%. Some of the

Polyglycine hydrolases secreted by pathogenic fungi

USDA-ARS?s Scientific Manuscript database

Pathogens are known to produce proteases that target host defense proteins. Here we describe polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were puri...

Variants of glycoside hydrolases

DOEpatents

Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

2013-02-26

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Variants of glycoside hydrolases

DOEpatents

Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

2011-04-26

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Variants of glycoside hydrolases

DOEpatents

Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

2017-07-11

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Method for purifying bidentate organophosphorus compounds

DOEpatents

Schulz, Wallace W.

1977-01-01

Bidentate organophosphorus compounds useful for extracting actinide elements from acidic nuclear waste solutions are purified of undesirable acidic impurities by contacting the compounds with ethylene glycol which preferentially extracts the impurities found in technical grade bidentate compounds.

Hydrolases of Hysterothylacium aduncum (Nematoda).

PubMed

Zółtowska, Krystyna; Dmitryjuk, Małgorzata; Rokicki, Jerzy; Lopieńska-Biernat, Elzbieta

2007-01-01

Enzymatic activity is an indicator of an organism's metabolic rate which depends on, i.e., environmental conditions, developmental stage, physiological stage, and sex. The API ZYM test was applied to compare activities of 19 hydrolases of female and male Hysterothylacium aduncum. Sexually mature nematodes were isolated from eelpout individuals caught in the Gulf of Gdańsk. Enzymatic activity of the hydrolases and the protein content was determined in nematode extracts using API ZYM and Bradford's method, respectively. The females and males tested showed a total of 13 enzymes to be active. The males showed additionally the presence of alpha-fucosidase. Acidic and alkaline phosphatases had very high activities in both sexes; short-chain fatty acid esterases, leucine and valine aminopeptidases, alpha-glucosidase, and N-acetylglucosaminidase were highly active. H. aduncum showed no trypsin- and chymotrypsin-specific activities; similarly, no activity of alpha-galactosidase, alpha-mannosidase, and beta-glucuronidase was revealed. Except for lipase (C14), hydrolases were more active in females than in males, which is related to metabolic rate being higher in females due to their reproductive function. Comparison of the results obtained with earlier data produced with API ZYM allowed suggesting that the hydrolase pattern may be more affected by habitat in the host than by the taxonomic affiliation of nematode.

Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P{submore » R} and P{sub S} stereoisomers at the P-chiral center. The tabun complex displayed only the P{sub R} stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.« less

Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes.

PubMed

Samanta, Uttamkumar; Kirby, Stephen D; Srinivasan, Prabhavathi; Cerasoli, Douglas M; Bahnson, Brian J

2009-08-15

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.

Organophosphorus and carbamate pesticides. Chapter 12 in Handbook of Ecotoxicology

USGS Publications Warehouse

Hill, Elwood F.; Hoffman, David J.; Rattner, Barnett A.; Burton, G. Allen; Cairns, John

1995-01-01

Organophosphorus and carbamate pesticides are used throughout the world to control a large variety of insects and other invertebrates, fungi, birds, mammals, and herbaceous plants. Over 100 different organophosphorus and carbamate chemicals are registered in the U.S. alone for use in thousands of products applied to widely diverse habitats including agricultural crops, forests, rangelands, wetlands, towns, and cities. These applications are estimated to be nearly 200 million acre-treatments (i.e., number of acres treated corrected for number of treatments) per year to control nuisance, depradating, and disease-bearing invertebrates and vertebrates, and to maintain landscape aesthetics. Except for mosquito control, most applications target terrestrial habitat. Due to drift or run-off, pesticide and degrades are inevitably detected in soils and water that are fundamental to the primary productivity of ecosystems. Thus, critical life-giving systems are frequently contaminated with organophosphorus and carbamate pesticides, however briefly, each year.

Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study.

PubMed

Eddleston, Michael; Eyer, Peter; Worek, Franz; Mohamed, Fahim; Senarathna, Lalith; von Meyer, Ludwig; Juszczak, Edmund; Hittarage, Ariyasena; Azhar, Shifa; Dissanayake, Wasantha; Sheriff, M H Rezvi; Szinicz, Ladislaus; Dawson, Andrew H; Buckley, Nick A

Although more than 100 organophosphorus insecticides exist, organophosphorus poisoning is usually regarded as a single entity, distinguished only by the compound's lethal dose in animals. We aimed to determine whether the three most common organophosphorus insecticides used for self-poisoning in Sri Lanka differ in the clinical features and severity of poisoning they cause. We prospectively studied 802 patients with chlorpyrifos, dimethoate, or fenthion self-poisoning admitted to three hospitals. Blood cholinesterase activity and insecticide concentration were measured to determine the compound and the patients' response to insecticide and therapy. We recorded clinical outcomes for each patient. Compared with chlorpyrifos (35 of 439, 8.0%), the proportion dying was significantly higher with dimethoate (61 of 264, 23.1%, odds ratio [OR] 3.5, 95% CI 2.2-5.4) or fenthion (16 of 99, 16.2%, OR 2.2, 1.2-4.2), as was the proportion requiring endotracheal intubation (66 of 439 for chlorpyrifos, 15.0%; 93 of 264 for dimethoate, 35.2%, OR 3.1, 2.1-4.4; 31 of 99 for fenthion, 31.3%, 2.6, 1.6-4.2). Dimethoate-poisoned patients died sooner than those ingesting other pesticides and often from hypotensive shock. Fenthion poisoning initially caused few symptoms but many patients subsequently required intubation. Acetylcholinesterase inhibited by fenthion or dimethoate responded poorly to pralidoxime treatment compared with chlorpyrifos-inhibited acetylcholinesterase. Organophosphorus insecticide poisoning is not a single entity, with substantial variability in clinical course, response to oximes, and outcome. Animal toxicity does not predict human toxicity since, although chlorpyrifos is generally the most toxic in rats, it is least toxic in people. Each organophosphorus insecticide should be considered as an individual poison and, consequently, patients might benefit from management protocols developed for particular organophosphorus insecticides.

CUMULATIVE RISK ANALYSIS FOR ORGANOPHOSPHORUS PESTICIDES

EPA Science Inventory

Cumulative Risk Analysis for Organophosphorus Pesticides
R. Woodrow Setzer, Jr. NHEERL MD-74, USEPA, RTP, NC 27711

The US EPA has recently completed a risk assessment of the effects of exposure to 33 organophosphorous pesticides (OPs) through the diet, water, and resi...

Biodegradation of phthalic acid esters (PAEs) and in silico structural characterization of mono-2-ethylhexyl phthalate (MEHP) hydrolase on the basis of close structural homolog.

PubMed

Singh, Neha; Dalal, Vikram; Mahto, Jai Krishna; Kumar, Pravindra

2017-09-15

Three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 were isolated and characterized for their degradative potential. These strains efficiently degraded 77.4%-84.4% of DMP, 75.0%-75.7% of DEP and 71.7%-74.7% of DEHP, initial amount of each phthalate is 500mgL -1 of each phthalate, after 44h of incubation. GC-MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA. The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as MEHP, mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3Å, which is similar to the ES complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of PMEs to phthalate. Copyright © 2017 Elsevier B.V. All rights reserved.

N-acetylcysteine in Acute Organophosphorus Pesticide Poisoning: A Randomized, Clinical Trial.

PubMed

El-Ebiary, Ahmad A; Elsharkawy, Rasha E; Soliman, Nema A; Soliman, Mohammed A; Hashem, Ahmed A

2016-08-01

Organophosphorus poisoning is a major global health problem with hundreds of thousands of deaths each year. Research interest in N-acetylcysteine has grown among increasing evidence of the role of oxidative stress in organophosphorus poisoning. We aimed to assess the safety and efficacy of N-acetylcysteine as an adjuvant treatment in patients with acute organophosphorus poisoning. This was a randomized, controlled, parallel-group trial on 30 patients suffering from acute organophosphorus poisoning, who were admitted to the Poison Control Center of Tanta University Emergency Hospital, Tanta, Egypt, between April and September 2014. Interventions included oral N-acetylcysteine (600 mg three times daily for 3 days) as an added treatment to the conventional measures versus only the conventional treatment. Outcome measures included mortality, total dose of atropine administered, duration of hospitalization and the need for ICU admission and/or mechanical ventilation. A total of 46 patients were screened and 30 were randomized. No significant difference was found between both groups regarding demographic characteristics and the nature or severity of baseline clinical manifestations. No major adverse effects to N-acetylcysteine therapy were reported. Malondialdehyde significantly decreased and reduced glutathione significantly increased only in the NAC-treated patients. The patients on NAC therapy required less atropine doses than those who received only the conventional treatment; however, the length of hospital stay showed no significant difference between both groups. The study concluded that the use of N-acetylcysteine as an added treatment was apparently safe, and it reduced atropine requirements in patients with acute organophosphorus pesticide poisoning. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

PubMed Central

Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

2017-01-01

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

Mechanism-based inhibition of HsaD: a C-C bond hydrolase essential for survival of Mycobacterium tuberculosis in macrophage.

PubMed

Ryan, Ali; Keany, Sebastian; Eleftheriadou, Olga; Ballet, Romain; Cheng, Hung-Yuan; Sim, Edith

2014-01-01

Mycobacterium tuberculosis remains the leading cause of death by a bacterial pathogen worldwide. Increasing prevalence of multidrug-resistant organisms means prioritizing identification of targets for antituberculars. 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD), part of the cholesterol metabolism operon, is vital for survival within macrophage. The C-C bond hydrolase, HsaD, has a serine protease-like catalytic triad. We tested a range of serine protease and esterase inhibitors for their effects on HsaD activity. As well as providing a potential starting point for drug development, the data provides evidence for the mechanism of C-C bond hydrolysis. This screen also provides a route to initiate development of fragment-based inhibitors. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Structure–efficiency relationships of cyclodextrin scavengers in the hydrolytic degradation of organophosphorus compounds

PubMed Central

Letort, Sophie; Bosco, Michaël; Cornelio, Benedetta; Brégier, Frédérique; Daulon, Sébastien; Gouhier, Géraldine

2017-01-01

New derivatives of cyclodextrins were prepared in order to determine the relative importance of the structural key elements involved in the degradation of organophosphorus nerve agents. To avoid a competitive inclusion between the organophosphorus substrate and the iodosobenzoate group, responsible for its degradation, the latter group had to be covalently bound to the cyclodextrin scaffold. Although the presence of the α nucleophile iodosobenzoate was a determinant in the hydrolysis process, an imidazole group was added to get a synergistic effect towards the degradation of the agents. The degradation efficiency was found to be dependent on the relative position of the heterocycle towards the reactive group as well as on the nature of the organophosphorus derivative. PMID:28382180

Structure-efficiency relationships of cyclodextrin scavengers in the hydrolytic degradation of organophosphorus compounds.

PubMed

Letort, Sophie; Bosco, Michaël; Cornelio, Benedetta; Brégier, Frédérique; Daulon, Sébastien; Gouhier, Géraldine; Estour, François

2017-01-01

New derivatives of cyclodextrins were prepared in order to determine the relative importance of the structural key elements involved in the degradation of organophosphorus nerve agents. To avoid a competitive inclusion between the organophosphorus substrate and the iodosobenzoate group, responsible for its degradation, the latter group had to be covalently bound to the cyclodextrin scaffold. Although the presence of the α nucleophile iodosobenzoate was a determinant in the hydrolysis process, an imidazole group was added to get a synergistic effect towards the degradation of the agents. The degradation efficiency was found to be dependent on the relative position of the heterocycle towards the reactive group as well as on the nature of the organophosphorus derivative.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

Inhibition of polygylcine hydrolases by substrate analog peptides

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases are proteases secreted by fungal pathogens that target corn defense chitinases. They cleave interdomain glycine-glycine bonds within a polyglycine linker, separating substrate chitinases into two single domain proteins. Polyglycine hydrolases consist of 640 amino acids with a ...

CUMULATIVE EFFECTS OF ORGANOPHOSPHORUS OR CARBAMATE PESTICIDES.

EPA Science Inventory

This book chapter strives to summarize the body of literature exploring the toxic interaction of organophosphorus and carbamate pesticides in mixtures. This review represents one of the only reviews of the subject that has been published within the last 20 years. Specifically, th...

DETERMINATION OF ORGANOPHOSPHORUS COMPOUNDS BY GC-ICPMS

EPA Science Inventory

Accidental or intentional release of neurotoxic organophosphorus (OP) pesticides and OP chemical warfare agents (CWAs) are potential threats to public health and the environment. Such a release could involve any number of a large suite of OP chemicals. These compounds, as well a...

Crystal structure of bile salt hydrolase from Lactobacillus salivarius.

PubMed

Xu, Fuzhou; Guo, Fangfang; Hu, Xiao Jian; Lin, Jun

2016-05-01

Bile salt hydrolase (BSH) is a gut-bacterial enzyme that negatively influences host fat digestion and energy harvesting. The BSH enzyme activity functions as a gateway reaction in the small intestine by the deconjugation of glycine-conjugated or taurine-conjugated bile acids. Extensive gut-microbiota studies have suggested that BSH is a key mechanistic microbiome target for the development of novel non-antibiotic food additives to improve animal feed production and for the design of new measures to control obesity in humans. However, research on BSH is still in its infancy, particularly in terms of the structural basis of BSH function, which has hampered the development of BSH-based strategies for improving human and animal health. As an initial step towards the structure-function analysis of BSH, C-terminally His-tagged BSH from Lactobacillus salivarius NRRL B-30514 was crystallized in this study. The 1.90 Å resolution crystal structure of L. salivarius BSH was determined by molecular replacement using the structure of Clostridium perfringens BSH as a starting model. It revealed this BSH to be a member of the N-terminal nucleophile hydrolase superfamily. Crystals of apo BSH belonged to space group P21212, with unit-cell parameters a = 90.79, b = 87.35, c = 86.76 Å (PDB entry 5hke). Two BSH molecules packed perfectly as a dimer in one asymmetric unit. Comparative structural analysis of L. salivarius BSH also identified potential residues that contribute to catalysis and substrate specificity.

Detection and determination of organophosphorus insecticides in tissues by thin-layer chromatography.

PubMed

Tewari, S N; Harpalani, S P

1977-01-11

The toxicological analysis of 12 common organophosphorus insecticides is described. Suitable methods for the extraction of organophosphorus insecticides from tissues are proposed. The detection, identification and estimation of these insecticides by thin-layer chromatography is described for 25 solvent systems and a series of chromogenic reagents. The distribution of insecticides in human body tissues in five cases of poisoning by ethyl parathion, malathion, dimethoate, sumithion and phosphamidon has also been studied.

Influence of N-P base fiber reactive organophosphorus flame retardant on cotton thermal behavior

USDA-ARS?s Scientific Manuscript database

An efficient synergistic effect between a nitrogen-containing organophosphorus compound in the presence of a catalytic amount of chlorine is proposed based on the cyanuric chloride-linked organophosphorus flame retardant, tetraethyl-2,2'-(6-chloro-1,3,5-triazine-2,4-diyl)bis(azanediyl)bis(ethane-2,1...

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

PubMed Central

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

Acute organo-phosphorus pesticide poisoning in North Karnataka, India: oxidative damage, haemoglobin level and total leukocyte.

PubMed

Hundekari, I A; Suryakar, A N; Rathi, D B

2013-03-01

Pesticide poisoning is an important cause of morbidity and mortality in India. To assess the oxidative damage, hemoglobin level and leukocyte count in acute organophosphorus pesticide poisoning. Plasma cholinesterase was assessed as a toxicity marker. Oxidative damage was assessed by estimating serum malondialdehyde (MDA) levels, plasma total antioxidant capacity (TAC), erythrocyte superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels. Progressive and significant decline (p< 0.001) in plasma cholinesterase in correlation with the severity of organophosphorus poisoning was observed. Serum MDA levels significantly increased (p< 0.001) in all grades of organophosphorus poisoning cases as compared to controls. Erythrocyte SOD, CAT and GPx were significantly increased (p< 0.05) in earlier grade and (p< 0.001) in later grades of organophosphorus poisoning cases as compared to controls. While plasma TAC (p<0.001) was significantly decreased in all grades of organophosphorus poisoning cases as compared to controls. Leucocytosis observed in these cases signifies the activation of defense mechanism which could be a positive response for survival. Organophosphorus compounds inhibit cholinesterase action leading to cholinergic hyperactivity. Increased MDA level may lead to peroxidative damages deteriorating the structural and functional integrity of neuronal membrane. Increased erythrocyte SOD, CAT and GPx activities suggest an adaptive measure to tackle the pesticide accumulation. Hence it is concluded that cholinesterase inhibition may initiate cellular dysfunction leading to acetylcholine induced oxidative damage.

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Higgins, M.; Whitworth, G; El Warry, N

2009-01-01

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in themore » other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.« less

Antimicrobial Activity of Galangin and Its Effects on Murein Hydrolases of Vancomycin-Intermediate Staphylococcus aureus (VISA) Strain Mu50.

PubMed

Ouyang, Jing; Sun, Fengjun; Feng, Wei; Xie, Yonghong; Ren, Lijuan; Chen, Yongchuan

2018-01-01

Backgroud: Antibiotic treatment for infections caused by vancomycin-intermediate Staphylococcus aureus (VISA) strains is challenging, and only a few effective and curative methods have been developed to combat these strains. This study aimed to investigate the antimicrobial activity of galangin against S. aureus and its effects on the murein hydrolases of VISA strain Mu50. This is the first report on these effects of galangin, and it may help to improve the treatment for VISA infections by demonstrating the effective use of galangin. Firstly, the minimum inhibitory concentration (MIC) and growth curve were used to investigate the antimicrobial activity of galangin against S. aureus. Secondly, transmission electron microscopy (TEM) was used to observe morphological changes of VISA strain Mu50. Thirdly, Triton X-100-induced autolysis and cell wall hydrolysis assays were performed to determine the activities of the murein hydrolases of Mu50. Finally, fluorescence real-time quantitative PCR was used to investigate the expression of the murein hydrolase-related Mu50 genes. The results indicated that the MIC of galangin was 32 μg/mL against ATCC25293, N315, and Mu50, and galangin could significantly suppress the bacterial growth (p < 0.05) with concentrations of 4, 8 and 16 μg/mL, compared with control group (0 μg/mL). To explore the possible reasons of bacteriostatic effects of galangin, we observed morphological changes using TEM which showed that the division of Mu50 daughter cells treated with galangin was obviously inhibited. Considering the vital role of murein hydrolases in cellular division, assays were performed, and galangin markedly decreased Triton X-100-induced autolysis and cell wall hydrolysis. Galangin also significantly inhibited the expression of the murein hydrolase genes (atl, lytM, and lytN) and their regulatory genes (cidR, cidA, and cidB). Our findings indicated that galangin can effectively inhibit murein hydrolase activity as well as the

Different approaches to acute organophosphorus poison treatment.

PubMed

Nurulain, Syed Muhammad

2012-07-01

Organophosphorus compounds (OPCs) have a wide variety of applications and are a serious threat for self-poisoning, unintentional misuse, terrorist attack, occupational hazard and warfare attack. The present standard treatment has been reported to be unsatisfactory. Many novel approaches are being used and tested for acute organophosphorus (OP) poison treatment. The bioscavenger concept captured high attention among the scientific community during the last few decades. Other approaches like alkalinisation of blood plasma/serum and use of weak inhibitors against strong inhibitors, though it showed promising results, did not get such wide attention. The introduction of a novel broad-spectrum oxime has also been in focus. In this mini-review, an update of the overview of four different approaches has been discussed. The standard therapy that is atropine+oxime+benzodiazepine along with supportive measures will continue to be the best option with only the replacement of a single oxime to improve its broad-spectrum efficacy.

Development of the Electrochemical Biosensor for Organophosphate Chemicals Using CNT/Ionic Liquid Bucky Gel Electrode

DTIC Science & Technology

2010-04-01

www.elsevier .com/locate /e lecomDevelopment of the electrochemical biosensor for organophosphate chemicals using CNT/ ionic liquid bucky gel electrode Bong...hydrolase Ionic liquid CNT Electrochemical property1388-2481/$ - see front matter 2009 Elsevier B.V. A doi:10.1016/j.elecom.2009.01.006 * Corresponding...kaist.ac.kr (S.Y. Lee), whhOrganophosphorus hydrolase (OPH) immobilized on CNT/ ionic liquid (IL) electrodes were prepared by using three different intrinsic

Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase.

PubMed

Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

2016-02-01

Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting.

Sorption of Organophosphorus Flame Retardants on Settled Dust

EPA Science Inventory

Organophosphorus flame retardants (OPFRs) are widely used as additives in industrial and consumer products such as electrical and electronic products, furniture, plastics, textiles, and building/construction materials. Due to human exposure and potential health effects, OPFRs inc...

Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyler, Ludmila; Bragg, Jennifer; Wu, Jiajie

2010-01-01

Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice), the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolasemore » genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model) and Sorghum bicolor (sorghum). We then compared the glycoside hydrolases across species, both at the whole-genome level and at the level of individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. Examination of individual glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51) revealed both similarities and distinctions between monocots and dicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets the stage for Brachypodium to be a monocot model for investigations of these enzymes and their diverse roles in planta

Sorption of Organophosphorus Flame-Retardants on Settled Dust

EPA Science Inventory

Dust is an important sink for indoor air pollutants, such as organophosphorus flame-retardants (OPFRs) that are used as additives in industrial and consumer products including electrical and electronic products, furniture, plastics, textile, and building/construction materials. T...

Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

PubMed

Hiraishi, Tomohiro

2016-02-01

Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

ORGANOPHOSPHORUS PESTICIDE DEGRADATION PATHWAYS DURING DRINKING WATER TREATMENT

EPA Science Inventory

The objective of this work was to investigate organophosphorus (OP) pesticide transformation pathways as a class in the presence of aqueous chlorine. Seven priority OP pesticides were examined for their reactivity with aqueous chlorine: chlorpyrifos (CP), parathion (PA), diazino...

Kinetic parameters of cholinesterase interactions with organophosphates: retrieval and comparison tools available through ESTHER database: ESTerases, alpha/beta Hydrolase Enzymes and Relatives.

PubMed

Chatonnet, A; Hotelier, T; Cousin, X

1999-05-14

Cholinesterases are targets for organophosphorus compounds which are used as insecticides, chemical warfare agents and drugs for the treatment of disease such as glaucoma, or parasitic infections. The widespread use of these chemicals explains the growing of this area of research and the ever increasing number of sequences, structures, or biochemical data available. Future advances will depend upon effective management of existing information as well as upon creation of new knowledge. The ESTHER database goal is to facilitate retrieval and comparison of data about structure and function of proteins presenting the alpha/beta hydrolase fold. Protein engineering and in vitro production of enzymes allow direct comparison of biochemical parameters. Kinetic parameters of enzymatic reactions are now included in the database. These parameters can be searched and compared with a table construction tool. ESTHER can be reached through internet (http://www.ensam.inra.fr/cholinesterase). The full database or the specialised X-window Client-server system can be downloaded from our ftp server (ftp://ftp.toulouse.inra.fr./pub/esther). Forms can be used to send updates or corrections directly from the web.

Glycemic Status in Organophosphorus Poisoning.

PubMed

Panda, S; Nanda, R; Mangaraj, M; Rathod, P K; Mishra, P K

2015-01-01

Organophosphorus(OP) poisoning, in addition to its cholinergic manifestations shows metabolic derangements leading to hyperglycemia. Apart from inhibiting acetylcholinesterase it also induces oxidative stress to exhibit this manifestation. The present study aims to assess the glycemic status of OP poisoned patients and its association with various factors in OP poisoning like oxidative stress and dose of atropine. This is a prospective study which recruited 102 patients above 18 years of age with history of OP poisoning. They were categorized into 3 grades-mild, moderate and severe based on the Peradeniya Organophosphorus Poisining Scale. The routine biochemical parameters along with serum malondialdehyde (MDA) and cholinesterase were estimated in the study group. Hyperglycemia and glycosuria were observed, with majority cases of hyperglycemia (57%) noticed in the severe group. There was a rise in the random plasma glucose (RPG), serum malondialdehyde (MDA), total dose of atropine across the groups along with a fall in the serum cholinesterase with increase in severity of poisoning. The fall in plasma glucose at the time of discharge was significant in all three groups when compared to the admission random plasma glucose(RPG) level. This transient hyperglycemia exhibited a significant positive association with serum MDA and dose of atropine administered during treatment (p<0.05). Glycemic status in OP poisoning may play a role in identifying the severity of poisoning at the time of admission.

MICROCHIP ENZYMATIC ASSAY OF ORGANOPHOSPHATE NERVE AGENTS. (R830900)

EPA Science Inventory

An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...

REMOTE BIOSENSOR FOR IN SITU MONITORING OF ORGANOPHOSPHATE NERVE AGENTS. (R823663)

EPA Science Inventory

A remote electrochemical biosensor for field monitoring of organophosphate nerve agents is described. The new sensor relies on the coupling of the effective biocatalytic action of organophosphorus hydrolase (OPH) with a submersible amperometric probe design. This combination resu...

Modulation of Immune Response by Organophosphorus Pesticides: Fishes as a Potential Model in Immunotoxicology

PubMed Central

Díaz-Resendiz, K. J. G.; Toledo-Ibarra, G. A.; Girón-Pérez, M. I.

2015-01-01

Immune response is modulated by different substances that are present in the environment. Nevertheless, some of these may cause an immunotoxic effect. In this paper, the effect of organophosphorus pesticides (frequent substances spilled in aquatic ecosystems) on the immune system of fishes and in immunotoxicology is reviewed. Furthermore, some cellular and molecular mechanisms that might be involved in immunoregulation mechanisms of organophosphorus pesticides are discussed. PMID:25973431

Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

PubMed Central

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-01-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

Toward antibody-catalyzed hydrolysis of organophosphorus poisons

PubMed Central

Vayron, Philippe; Renard, Pierre-Yves; Taran, Frédéric; Créminon, Christophe; Frobert, Yveline; Grassi, Jacques; Mioskowski, Charles

2000-01-01

We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-α-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-α-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M−1⋅min−1 at pH 7.4 and 25°C, which corresponds to a catalytic proficiency of 14,400 M−1 toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents. PMID:10860971

Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Read, David J.; Langford, Lynda; Barbour, Helen R.

2007-03-15

Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [{sup 14}C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [{sup 14}C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTEmore » inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [{sup 14}C]phosphatidylcholine to [{sup 14}C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with > 80% inhibition of [{sup 14}C]GroPCho production, increased [{sup 14}C]phosphatidylcholine was not detected. At concentrations of 1-10 {mu}M, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.« less

Chloramination of Organophosphorus Pesticides Found in Drinking Water Sources

EPA Science Inventory

The degradation of commonly detected organophosphorus (OP) pesticides, in drinking water sources, was investigated under simulated chloramination conditions. Due to monochloramine autodecomposition, it is difficult to observe the direct reaction of monochloramine with each OP pe...

Sorption of Organophosphorus Flame Retardants (OPFRs) on Settled Dust

EPA Science Inventory

Organophosphorus flame retardants (OPFRs) are widely used as additives in industrial and consumer products such as electrical and electronic products, furniture, plastics, textiles, and building/construction materials. Due to human exposure and potential health effects, OPFRs inc...

Acute organophosphorus poisoning complicated by acute coronary syndrome.

PubMed

Pankaj, Madhu; Krishna, Kavita

2014-07-01

We report a case of 30 year old alcoholic male admitted with vomiting, drowsiness, limb weakness and fasciculations after alleged history of consumption of 30 ml of chlorpyriphos insecticide. He had low serum cholinesterase levels. With standard treatment for organophosphorus poisoning (OPP), he improved gradually until day 5, when he developed neck and limb weakness and respiratory distress. This intermediate syndrome was treated with oximes, atropine and artificial ventilation. During treatment, his ECG showed fresh changes of ST elevation. High CPK & CPK-MB levels, septal hypokinesia on 2D echo suggested acute coronary syndrome. Coronary angiography was postponed due to his bedridden and obtunded status. The patient finally recovered fully by day 15 and was discharged. Acute coronary syndrome is a rare occurrence in OP poisoning. The present case thus emphasises the need for careful electrocardiographic and enzymatic monitoring of all patients of organophosphorus poisoning to prevent potential cardiac complication which can prove fatal.

Parenteral organophosphorus poisoning in a rural emergency department: a case report

PubMed Central

2013-01-01

Background Poisoning is a common presentation in the emergency department. Oral exposures to organophosphorus compounds are especially frequent in rural and agricultural regions of South Asia and throughout the developing world. Case presentation Here we report a case of deliberate self-harm with an organophosphorus pesticide via the relatively uncommon parenteral route. A young woman injected herself with chlorpyriphos. Although the cholinergic effects were mild, cellulitis and abscess development were noted as a result. Conclusion Resource limited agricultural countries like Nepal present health care workers with numerous challenges in poisoning management. This case represents a rare but potentially morbid method of agrochemical poison exposure. PMID:24321121

Particulate matter formation from photochemical degradation of organophosphorus pesticides

NASA Astrophysics Data System (ADS)

Borrás, E.; Ródenas, M.; Vera, T.; Muñoz, A.

2015-12-01

Several experiments were performed in the European Photo-reactor - EUPHORE - for studying aerosol formation from organophosphorus pesticides such as diazinon, chlorpyrifos, chlorpyrifos-methyl and pirimiphos-methyl. The mass concentration yields obtained (Y) were in the range 5 - 44% for the photo-oxidation reactions in the presence and the absence of NOx. These results confirm the importance of studying pesticides as significant precursors of atmospheric particulate matter due to the serious risks associated to them. The studies based on the use of EUPHORE photoreactor provide useful data about atmospheric degradation processes of organophosphorus pesticides to the atmosphere. Knowledge of the specific degradation products, including the formation of secondary particulate matter, could complete the assessment of their potential impact, since the formation of those degradation products plays a significant role in the atmospheric chemistry, global climate change, radiative force, and are related to health effects.

Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allgaier, M.; Reddy, A.; Park, J. I.

2009-11-15

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence datamore » from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.« less

Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, Amitha; Allgaier, Martin; Park, Joshua I.

2011-05-11

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence datamore » from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.« less

Biodegradation of chlorpyrifos by bacterial genus Pseudomonas.

PubMed

Gilani, Razia Alam; Rafique, Mazhar; Rehman, Abdul; Munis, Muhammad Farooq Hussain; Rehman, Shafiq Ur; Chaudhary, Hassan Javed

2016-02-01

Chlorpyrifos is an organophosphorus pesticide commonly used in agriculture. It is noxious to a variety of organisms that include living soil biota along with beneficial arthropods, fish, birds, humans, animals, and plants. Exposure to chlorpyrifos may cause detrimental effects as delayed seedling emergence, fruit deformities, and abnormal cell division. Contamination of chlorpyrifos has been found about 24 km from the site of its application. There are many physico-chemical and biological approaches to remove organophosphorus pesticides from the ecosystem, among them most promising is biodegradation. The 3,5,6-trichloro-2-pyridinol (TCP) and diethylthiophosphate (DETP) as primary products are made when chlorpyrifos is degraded by soil microorganisms which further break into nontoxic metabolites as CO(2), H(2)O, and NH(3). Pseudomonas is a diversified genus possessing a series of catabolic pathways and enzymes involved in pesticide degradation. Pseudomonas putida MAS-1 is reported to be more efficient in chlorpyrifos degradation by a rate of 90% in 24 h among Pseudomonas genus. The current review analyzed the comparative potential of bacterial species in Pseudomonas genus for degradation of chlorpyrifos thus, expressing an ecofriendly approach for the treatment of environmental contaminants like pesticides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of an organophosphorus pesticide on reproduction in the rat.

DOT National Transportation Integrated Search

1970-01-01

The toxic effects of organophosphorus insecticides are commonly ascribed to cholinesterase (CHE) inhibition. A search of the scientific literature revealed the well-established fact that a number of chlorinated pesticides adversely affect reproductio...

BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS. 1. POTENTIOMETRIC ENZYME ELECTRODE. (R823663)

EPA Science Inventory

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP)
nerve agents was developed. The basic element of this enzyme electrode was a pH electrode
modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking
OPH ...

The lid domain of the MCP hydrolase DxnB2 contributes to the reactivity towards recalcitrant PCB metabolites

PubMed Central

Yam, Katherine C.; Ghosh, Subhangi; Bolin, Jeffrey T.; Eltis, Lindsay D.

2013-01-01

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically-shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ESred, an intermediate possessing a bathochromically-shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-hydrolase core domain, as a determinant in oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme’s ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/β-hydrolases. PMID:23879719

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

PubMed

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-05-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duysen, Ellen G.; Lockridge, Oksana, E-mail: olockrid@unmc.edu

2011-09-01

The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher thanmore » the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase. - Highlights: > Acetylcholinesterase activity is induced by organophosphorus agents. > AChE induction is related to apoptosis. > Induction of AChE activity by OP is independent of BChE.« less

QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis. [quantitative structure-activity relationship, complete neglect of differential overlap

NASA Technical Reports Server (NTRS)

Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

1985-01-01

Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of cationic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR-primeP(O)X, where R and R-prime are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrolysis rate constant.

Organophosphorus pesticide poisonings in humans: determination of residues and metabolites in tissues and urine.

PubMed

Lores, E M; Bradway, D E; Moseman, R F

1978-01-01

The analyses of four organophosphorus pesticide poisoning cases, three of which resulted in death, are reported. The case histories of the subjects, along with the analysis of tissues, urine, and blood for the levels of pesticides and metabolites are given. The pesticides involved include dicrotophos, chlorpyrifos, malathion, and parathion. The methods of analysis were adapted from previously published methods that provide a very rapid means of identification of organophosphorus pesticides in the tissues or in the blood of poisoned patients.

Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis

PubMed Central

Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

2014-01-01

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites. DOI: http://dx.doi.org/10.7554/eLife.01990.001 PMID:24957336

FIBER-OPTIC BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS. (R823663)

EPA Science Inventory

A fiber-optic enzyme biosensor for the direct measurement of organophosphate nerve
agents was developed. The basic element of this biosensor is organophosphorus hydrolase
immobilized on a nylon membrane and attached to the common end of a bifurcated optical fiber
bundle....

Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass.

PubMed

Gladden, John M; Allgaier, Martin; Miller, Christopher S; Hazen, Terry C; VanderGheynst, Jean S; Hugenholtz, Philip; Simmons, Blake A; Singer, Steven W

2011-08-15

Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

Crystal structure of wild-type and mutant human Ap4A hydrolase.

PubMed

Ge, Honghua; Chen, Xiaofang; Yang, Weili; Niu, Liwen; Teng, Maikun

2013-03-01

Ap4A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17), an enzyme involved in a number of biological processes, is characterized as cleaving the polyphosphate chain at the fourth phosphate from the bound adenosine moiety. This paper presents the crystal structure of wild-type and E58A mutant human Ap4A hydrolase. Similar to the canonical Nudix fold, human Ap4A hydrolase shows the common αβα-sandwich architecture. Interestingly, two sulfate ions and one diphosphate coordinated with some conserved residues were observed in the active cleft, which affords a better understanding of a possible mode of substrate binding. Copyright © 2013 Elsevier Inc. All rights reserved.

SARS coronavirus protein 7a interacts with human Ap4A-hydrolase.

PubMed

Vasilenko, Natalia; Moshynskyy, Igor; Zakhartchouk, Alexander

2010-02-09

The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm.

A molecular model for the active site of S-adenosyl- l-homocysteine hydrolase

NASA Astrophysics Data System (ADS)

Yeh, Jerry C.; Borchardt, Ronald T.; Vedani, Angelo

1991-06-01

S-adenosyl- l-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1.), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD+. Based on the 2.0 Å-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction. We also studied a model of the complex of AdoHcy hydrolase with NAD+, as well as the ternary complexes of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.

Sorption of triazine and organophosphorus pesticides on soil and biochar

USDA-ARS?s Scientific Manuscript database

Although a large number of reports are available on sorption and degradation of triazine and organophosphorus pesticides in soils, systematic studies are lacking to directly compare and predict the fate of agrochemicals having different susceptibilities for hydrolysis and other degradation pathways....

Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.

PubMed

Reeves, T E; Wales, M E; Grimsley, J K; Li, P; Cerasoli, D M; Wild, J R

2008-06-01

Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.

The nodulation factor hydrolase of Medicago truncatula: characterization of an enzyme specifically cleaving rhizobial nodulation signals.

PubMed

Tian, Ye; Liu, Wei; Cai, Jie; Zhang, Lan-Yue; Wong, Kam-Bo; Feddermann, Nadja; Boller, Thomas; Xie, Zhi-Ping; Staehelin, Christian

2013-11-01

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs.

Human alpha beta hydrolase domain containing protein 11 and its yeast homolog are lipid hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arya, Madhuri; Srinivasan, Malathi; Rajasekharan, Ram

Mammalian alpha/beta hydrolase domain (ABHD) family of proteins have emerged as key regulators of lipid metabolism and are found to be associated with human diseases. Human α/β-hydrolase domain containing protein 11 (ABHD11) has recently been predicted as a potential biomarker for human lung adenocarcinoma. In silico analyses of the ABHD11 protein sequence revealed the presence of a conserved lipase motif GXSXG. However, the role of ABHD11 in lipid metabolism is not known. To understand the biological function of ABHD11, we heterologously expressed the human ABHD11 in budding yeast, Saccharomyces cerevisiae. In vivo [{sup 14}C]acetate labeling of cellular lipids in yeast cellsmore » overexpressing ABHD11 showed a decrease in triacylglycerol content. Overexpression of ABHD11 also alters the molecular species of triacylglycerol in yeast. Similar activity was observed in its yeast homolog, Ygr031w. The role of the conserved lipase motif in the hydrolase activity was proven by the mutation of all conserved amino acid residues of GXSXG motif. Collectively, our results demonstrate that human ABHD11 and its yeast homolog YGR031W have a pivotal role in the lipid metabolism. - Highlights: • Overexpression of ABHD11 protein and its yeast homolog Ygr031w cause a reduction in triacylglycerol levels in yeast. • The reduction in triacylglycerol is due to the presence of lipase motif GXSXG. • Overexpression of ABHD11 and Ygr031w alters the molecular species of triacylglycerol.« less

Recognition and cleavage of corn defense chitinases by fungal polyglycine hydrolases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn. Polyglycine hydrolases are novel proteins in terms of activity and sequence. The objective of the study is to und...

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

PubMed Central

Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

2014-01-01

ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

HPLC SEPARATION OF CHIRAL ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

EPA Science Inventory

High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) were obtained on polysaccharide chiral HPLC columns using an alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, dyfonate, fenamiphos, ...

UPTAKE AND PHYTOTRANSFORMATION OF ORGANOPHOSPHORUS PESTICIDES BY AXENICALLY CULTIVATED AQUATIC PLANTS

EPA Science Inventory

The uptake and phytotransformation of organophosphorus (OP) pesticides (malathion, demeton-S-methyl, and crufomate) was investigated in vitro using the axenically aquatic cultivated plants parrot feather (Myriophyllum aquaticum), duckweed (Spirodela oligorrhiza L.), and elodea (E...

Visual detection of organophosphorus pesticides represented by mathamidophos using Au nanoparticles as colorimetric probe.

PubMed

Li, Hongkun; Guo, Jiajia; Ping, Hong; Liu, Lurui; Zhang, Minwei; Guan, Fengrui; Sun, Chunyan; Zhang, Qian

2011-12-15

With citrate-coated Au nanoparticles as colorimetric probe, a novel visual method for rapid assay of organophosphorus pesticides has been developed. The assay principle is based on catalytic hydrolysis of acetylthiocholine into thiocholine by acetylcholinesterase, which induces the aggregation of Au nanoparticles and the color change from claret-red to purple or even grey. The original plasmon absorption of Au nanoparticles at 522 nm decreases, and simultaneously, a new absorption band appears at 675 nm. The irreversible inhibition of organophosphorus pesticides on acetylcholinesterase prevents aggregation of Au nanoparticles. Under optimum conditions, the absorbance at 522 nm of Au nanoparticles is related linearly to the concentration of mathamidophos in the range of 0.02-1.42 μg/mL with a detection limit of 1.40 ng/mL. This colorimetric method has been successfully utilized to detect mathamidophos in vegetables with satisfactory results. The proposed colorimetric assay exhibits good reproducibility and accuracy, providing a simple and rapid method for the analysis of organophosphorus pesticides. Copyright © 2011 Elsevier B.V. All rights reserved.

Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides.

PubMed

Long, Qian; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

2015-06-15

This paper reports a novel nanosensor for organophosphorus pesticides based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The detection mechanism is based on the facts that AuNPs quench the fluorescence of UCNPs and organophosphorus pesticides (OPs) inhibit the activity of acetylcholinesterase (AChE) which catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine. Under the optimized conditions, the logarithm of the pesticides concentration was proportional to the inhibition efficiency. The detection limits of parathion-methyl, monocrotophos and dimethoate reached 0.67, 23, and 67 ng/L, respectively. Meanwhile, the biosensor shows good sensitivity, stability, and could be successfully applied to detection of OPs in real food samples, suggesting the biosensor has potentially extensive application clinic diagnoses assays. Copyright © 2014 Elsevier B.V. All rights reserved.

Legionella pneumophila NudA Is a Nudix hydrolase and virulence factor.

PubMed

Edelstein, Paul H; Hu, Baofeng; Shinzato, Takashi; Edelstein, Martha A C; Xu, Wenlian; Bessman, Maurice J

2005-10-01

We studied the identity and function of the 528-bp gene immediately upstream of Legionella pneumophila F2310 ptsP (enzyme I(Ntr)). This gene, nudA, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as Escherichia coli YgdP, in that Ap(n)A's, in particular diadenosine pentaphosphate (Ap(5)A), were the preferred substrates. NudA hydrolyzed Ap(5)A to ATP plus ADP. Both ptsP and nudA were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene nudA was present in 19 of 20 different L. pneumophila strains tested and in 5 of 10 different Legionella spp. other than L. pneumophila. An in-frame nudA mutation was made in L. pneumophila F2310 to determine the phenotype. The nudA mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25 degrees C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with nudA. The nudA mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with L. pneumophila pneumonia showed that the nudA mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in L. pneumophila and is a virulence factor.

Benzil, a potent activator of microsomal epoxide hydrolase in vitro.

PubMed

Seidegård, J; DePierre, J W

1980-12-01

Benzil was found to be a very potent activator of microsomal epoxide hydrolase activity (measured with styrene oxide as substrate) in vitro. The activating effect was uncompetitive and benzil causes approximately ninefold increases in both the apparent V and the apparent Km of the enzyme(s). The half-maximal effect on activity was obtained as a 0.3 mM concentration of benzil. The activating effect obtained with benzil was found to be very specific, since a variety of structurally related compounds had little or no effect on microsomal epoxide hydrolase activity. In order to obtain indications for the existence of more than one microsomal epoxide hydrolase the effect of benzil on this activity from rats induced with phenobarbital, 3-methylcholanthrene, 2-acetylaminofluorene, trans-stilbene oxide, and benzil was tested. The differences observed were minor.

Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

PubMed Central

Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

2012-01-01

We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

Structure and function of polyglycine hydrolases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglyci...

Clinical analysis of penehyclidine hydrochloride combined with hemoperfusion in the treatment of acute severe organophosphorus pesticide poisoning.

PubMed

Liang, M J; Zhang, Y

2015-05-11

This study aimed to observe the clinical curative effect of penehyclidine hydrochloride (PHC) combined with hemoperfusion in treating acute severe organophosphorus pesticide poisoning. We randomly divided 61 patients with severe organophosphorus pesticide poisoning into an experimental group (N = 31) and a control group (N = 30), and we compared the coma-recovery time, mechanical ventilation time, healing time, hospital expenses, and mortality between the two groups. The coma-recovery time, mechanical ventilation time, and healing time were lower in the experimental group than in the control group (P < 0.05), while the hospitalization expenses were higher in the experimental group than in the control group (P < 0.01); moreover, no significant difference was observed in the mortality rate between the two groups. Thus, PHC combined with hemoperfusion exerts a better therapeutic effect in acute severe organophosphorus pesticide poisoning than PHC alone.

Natural diversity of glycoside hydrolase family 48 exoglucanases: insights from structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunecky, Roman; Alahuhta, Markus; Sammond, Deanne W.

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction. Surprisingly, given their importance for these microorganisms, GH48s have intrinsically low cellulolytic activity but even in low ratios synergize greatly with GH9 endoglucanases. In this study, we explore the structural and enzymatic diversity of these enzymes across a wide range of temperature optima. We have crystallized one new GH48 module from Bacillus pumilus in a complex withmore » cellobiose and cellohexaose (BpumGH48). We compare this structure to other known GH48 enzymes in an attempt to understand GH48 structure/function relationships and draw general rules correlating amino acid sequences and secondary structures to thermostability in this GH family.« less

Natural diversity of glycoside hydrolase family 48 exoglucanases: insights from structure

DOE PAGES

Brunecky, Roman; Alahuhta, Markus; Sammond, Deanne W.; ...

2017-11-30

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction. Surprisingly, given their importance for these microorganisms, GH48s have intrinsically low cellulolytic activity but even in low ratios synergize greatly with GH9 endoglucanases. In this study, we explore the structural and enzymatic diversity of these enzymes across a wide range of temperature optima. We have crystallized one new GH48 module from Bacillus pumilus in a complex withmore » cellobiose and cellohexaose (BpumGH48). We compare this structure to other known GH48 enzymes in an attempt to understand GH48 structure/function relationships and draw general rules correlating amino acid sequences and secondary structures to thermostability in this GH family.« less

Acyloxyacyl hydrolase promotes the resolution of lipopolysaccharide-induced acute lung injury

PubMed Central

Tang, Zihui; Yang, Qian; Qian, Guojun; Qian, Jing; Zeng, Wenjiao; Gu, Jie; Chu, Tianqing; Zhu, Ning; Zhang, Wenhong; Yan, Dapeng; He, Rui; Chu, Yiwei

2017-01-01

Pulmonary infection is the most common risk factor for acute lung injury (ALI). Innate immune responses induced by Microbe-Associated Molecular Pattern (MAMP) molecules are essential for lung defense but can lead to tissue injury. Little is known about how MAMP molecules are degraded in the lung or how MAMP degradation/inactivation helps prevent or ameliorate the harmful inflammation that produces ALI. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates Gram-negative bacterial endotoxin (lipopolysaccharide, or LPS). We report here that alveolar macrophages increase AOAH expression upon exposure to LPS and that Aoah+/+ mice recover more rapidly than do Aoah-/- mice from ALI induced by nasally instilled LPS or Klebsiella pneumoniae. Aoah-/- mouse lungs had more prolonged leukocyte infiltration, greater pro- and anti-inflammatory cytokine expression, and longer-lasting alveolar barrier damage. We also describe evidence that the persistently bioactive LPS in Aoah-/- alveoli can stimulate alveolar macrophages directly and epithelial cells indirectly to produce chemoattractants that recruit neutrophils to the lung and may prevent their clearance. Distinct from the prolonged tolerance observed in LPS-exposed Aoah-/- peritoneal macrophages, alveolar macrophages that lacked AOAH maintained or increased their responses to bioactive LPS and sustained inflammation. Inactivation of LPS by AOAH is a previously unappreciated mechanism for promoting resolution of pulmonary inflammation/injury induced by Gram-negative bacterial infection. PMID:28622363

Isolatable organophosphorus(III)-tellurium heterocycles.

PubMed

Nordheider, Andreas; Chivers, Tristram; Schön, Oliver; Karaghiosoff, Konstantin; Athukorala Arachchige, Kasun S; Slawin, Alexandra M Z; Woollins, J Derek

2014-01-13

A new structural arrangement Te3 (RP(III) )3 and the first crystal structures of organophosphorus(III)-tellurium heterocycles are presented. The heterocycles can be stabilized and structurally characterized by the appropriate choice of substituents in Tem (P(III) R)n (m=1: n=2, R=OMes* (Mes*=supermesityl or 2,4,6-tri-tert-butylphenyl); n=3, R=adamantyl (Ad); n=4, R=ferrocene (Fc); m=n=3: R=trityl (Trt), Mesor by the installation of a P(V) 2 N2 anchor in RP(III) [TeP(V) (tBuN)(μ-NtBu)]2 (R=Ad, tBu). Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of alpha lipoic acid on leukotriene A4 hydrolase.

PubMed

Torres, María José; Fierro, Angélica; Pessoa-Mahana, C David; Romero-Parra, Javier; Cabrera, Gonzalo; Faúndez, Mario

2017-03-15

Leukotriene A 4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A 4 to leukotriene B 4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B 4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B 4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A 4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A 4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A 4 hydrolase using in vivo models. Copyright © 2017 Elsevier B.V. All rights reserved.

A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16▿†

PubMed Central

Bers, Karolien; Leroy, Baptiste; Breugelmans, Philip; Albers, Pieter; Lavigne, Rob; Sørensen, Sebastian R.; Aamand, Jens; De Mot, René; Wattiez, Ruddy; Springael, Dirk

2011-01-01

The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria. PMID:22003008

Enzyme-based electrochemical biosensors for determination of organophosphorus and carbamate pesticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Everett, W.R.; Rechnitz, G.A.

1999-01-01

A mini review of enzyme-based electrochemical biosensors for inhibition analysis of organophosphorus and carbamate pesticides is presented. Discussion includes the most recent literature to present advances in detection limits, selectivity and real sample analysis. Recent reviews on the monitoring of pesticides and their residues suggest that the classical analytical techniques of gas and liquid chromatography are the most widely used methods of detection. These techniques, although very accurate in their determinations, can be quite time consuming and expensive and usually require extensive sample clean up and pro-concentration. For these and many other reasons, the classical techniques are very difficult tomore » adapt for field use. Numerous researchers, in the past decade, have developed and made improvements on biosensors for use in pesticide analysis. This mini review will focus on recent advances made in enzyme-based electrochemical biosensors for the determinations of organophosphorus and carbamate pesticides.« less

[A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A. royreba

PubMed

Kim, Yong Kyu; Kim, Tae Ue; Joung, In Sil; Im, Kyung Il

1988-06-01

Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta-N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80% of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba

Characterization of organophosphorus flame retardants' sorption on building materials and consumer products

EPA Science Inventory

Better understanding the transport mechanisms of organophosphorus flame-retardants (OPFRs) in the residential environment is important to more accurately estimate their indoor exposure and develop risk management strategies that protect human health. This study describes an impro...

Processing of Cholinesterase-like α/β-Hydrolase Fold Proteins: Alterations Associated with Congenital Disorders

PubMed Central

De Jaco, Antonella; Comoletti, Davide; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2016-01-01

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β-hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold. PMID:21933121

Integrated microwave processing system for the extraction of organophosphorus pesticides in fresh vegetables.

PubMed

Wu, Lijie; Song, Ying; Hu, Mingzhu; Xu, Xu; Zhang, Hanqi; Yu, Aimin; Ma, Qiang; Wang, Ziming

2015-03-01

A simple and efficient integrated microwave processing system (IMPS) was firstly assembled and validated for the extraction of organophosphorus pesticides in fresh vegetables. Two processes under microwave irradiation, dynamic microwave-assisted extraction (DMAE) and microwave-accelerated solvent elution (MASE), were integrated for simplifying the pretreatment of the sample. Extraction, separation, enrichment and elution were finished in a simple step. The organophosphorus pesticides were extracted from the fresh vegetables into hexane with DMAE, and then the extract was directly introduced into the enrichment column packed with active carbon fiber (ACF). Subsequently, the organophosphorus pesticides trapped on the ACF were eluted with ethyl acetate under microwave irradiation. No further filtration or cleanup was required before analysis of the eluate by gas chromatography-mass spectrometry. Some experimental parameters affecting extraction efficiency were investigated and optimized, such as microwave output power, kind and volume of extraction solvent, extraction time, amount of sorbent, elution microwave power, kind and volume of elution solvent, elution solvent flow rate. Under the optimized conditions, the recoveries were in the range of 71.5-105.2%, and the relative standard deviations were lower than 11.6%. The experiment results prove that the present method is a simple and effective sample preparation method for the determination of pesticides in solid samples. Copyright © 2014 Elsevier B.V. All rights reserved.

A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations

PubMed Central

Lodola, Alessio; Branduardi, Davide; De Vivo, Marco; Capoferri, Luigi; Mor, Marco; Piomelli, Daniele; Cavalli, Andrea

2012-01-01

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors. PMID:22389698

S-adenosylhomocysteine hydrolase deficiency in a 26-year-old man.

PubMed

Buist, N R M; Glenn, B; Vugrek, O; Wagner, C; Stabler, S; Allen, R H; Pogribny, I; Schulze, A; Zeisel, S H; Barić, I; Mudd, S H

2006-08-01

This paper reports the third proven human case of deficient S-adenosylhomocysteine (AdoHcy) hydrolase activity. The patient is similar to the only two previously reported cases with this disorder in having severe myopathy, developmental delay, elevated serum creatine kinase (CK) concentrations, and hypermethioninaemia. Although he has been followed from infancy, the basic enzyme deficiency was established only at age 26 years. The diagnosis was based on markedly elevated plasma concentrations of both AdoHcy and S-adenosylmethionine, some 20% of the mean control activity of AdoHcy hydrolase activity in haemolysates of his red-blood cells, and two missense mutations in his gene encoding AdoHcy hydrolase. He had low values of erythrocyte phosphatidylcholine and plasma free choline and marginally elevated excretion of guanidinoacetate, suggesting that the elevated AdoHcy may have been inhibiting methylation of phosphatidylethanolamine and guanidinoacetate. His leukocyte DNA was globally more methylated than the DNA's of his parents or the mean extent of methylation measured in age-matched control subjects.

The Nodulation Factor Hydrolase of Medicago truncatula: Characterization of an Enzyme Specifically Cleaving Rhizobial Nodulation Signals1[W][OPEN

PubMed Central

Tian, Ye; Liu, Wei; Cai, Jie; Zhang, Lan-Yue; Wong, Kam-Bo; Feddermann, Nadja; Boller, Thomas; Xie, Zhi-Ping; Staehelin, Christian

2013-01-01

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs. PMID:24082029

Functional metagenomics of oil-impacted mangrove sediments reveals high abundance of hydrolases of biotechnological interest.

PubMed

Ottoni, Júlia Ronzella; Cabral, Lucélia; de Sousa, Sanderson Tarciso Pereira; Júnior, Gileno Vieira Lacerda; Domingos, Daniela Ferreira; Soares Junior, Fábio Lino; da Silva, Mylenne Calciolari Pinheiro; Marcon, Joelma; Dias, Armando Cavalcante Franco; de Melo, Itamar Soares; de Souza, Anete Pereira; Andreote, Fernando Dini; de Oliveira, Valéria Maia

2017-07-01

Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms. For this, an in-depth survey of the taxonomic and functional microbial diversity recovered in a fosmid library (Library_Oil Mgv) constructed from oil-impacted Brazilian mangrove sediment was carried out. Fosmid DNA of the whole library was extracted and submitted to Illumina HiSeq sequencing. The resulting Library Oil_Mgv dataset was further compared with those obtained by direct sequencing of environmental DNA from Brazilian mangroves (from distinct regions and affected by distinct sources of contamination), focusing on hydrolases with potential use in biotechnological processes. The most abundant hydrolases found were proteases, esterases and amylases, with similar occurrence profile in all datasets. The main microbial groups harboring such hydrolase-encoding genes were distinct in each mangrove, and in the fosmid library these enzymes were mainly assigned to Chloroflexaceae (for amylases), Planctomycetaceae (for esterases) and Bradyrhizobiaceae (for proteases). Assembly and analysis of Library_Oil Mgv reads revealed three potentially novel enzymes, one epoxide hydrolase, one xylanase and one amylase, to be further investigated via heterologous expression assays.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

PubMed

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Biosynthesis of (R)-phenyl-1,2-ethanediol from racemic styrene oxide by using bacterial and marine fish epoxide hydrolases.

PubMed

Kim, Hee Sook; Lee, Ok Kyung; Hwang, Seungha; Kim, Beum Jun; Lee, Eun Yeol

2008-01-01

Enantio-convergent hydrolysis of racemic styrene oxides was achieved to prepare enantiopure (R)-phenyl-1,2-ethanediol by using two recombinant epoxide hydrolases (EHs) of a bacterium, Caulobacter crescentus, and a marine fish, Mugil cephalus. The recombinant C. crescentus EH primarily attacked the benzylic carbon of (S)-styrene oxide, while the M. cephalus EH preferentially attacked the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-Phenyl-1,2-ethanediol was obtained with 90% enantiomeric excess and yield as high as 94% from 50 mM racemic styrene oxides in a one-pot process.

Function of xyloglucan endotransglucosylase/hydrolases in rice

PubMed Central

Hara, Yoshinao; Yokoyama, Ryusuke; Osakabe, Keishi; Toki, Seiichi; Nishitani, Kazuhiko

2014-01-01

Background and Aims Although xyloglucans are ubiquitous in land plants, they are less abundant in Poales species than in eudicotyledons. Poales cell walls contain higher levels of β-1,3/1,4 mixed-linked glucans and arabinoxylans than xyloglucans. Despite the relatively low level of xyloglucans in Poales, the xyloglucan endotransglucosylase/hydrolase (XTH) gene family in rice (Oryza sativa) is comparable in size to that of the eudicotyledon Arabidopsis thaliana. This raises the question of whether xyloglucan is a substrate for rice XTH gene products, whose enzyme activity remains largely uncharacterized. Methods This study focused on OsXTH19 (which belongs to Group IIIA of the XTH family and is specifically expressed in growing tissues of rice shoots), and two other XTHs, OsXTH11 (Group I/II) and OsXTH20 (Group IIIA), for reference, and measurements were made of the enzymatic activities of three recombinant rice XTHs, i.e. OsXTH11, OsXTH20 and OsXTH19. Key Results All three OsXTH gene products have xyloglucan endohydrolase (XEH, EC 3·2·1·151) activity, and OsXTH11 has both XEH and xyloglucan endotransglycosylase (XET, EC 2·4·1207) activities. However, these proteins had neither hydrolase nor transglucosylase activity when glucuronoarabinoxylan or mixed-linkage glucan was used as the substrate. These results are consistent with histological observations demonstrating that pOsXTH19::GUS is expressed specifically in the vicinity of tissues where xyloglucan immunoreactivity is present. Transgenic rice lines over-expressing OsXTH19 (harbouring a Cauliflower Mosaic Virus 35S promoter::OsXTH19 cDNA construct) or with suppressed OsXTH19 expression (harbouring a pOsXTH19 RNAi construct) did not show dramatic phenotypic changes, suggesting functional redundancy and collaboration among XTH family members, as was observed in A. thaliana. Conclusions OsXTH20 and OsXTH19 act as hydrolases exclusively on xyloglucan, while OsXTH11 exhibits both hydrolase and XET activities

Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe.

PubMed

Robinson, A K; de la Peña, C E; Barnes, L D

1993-02-13

An enzyme that catalyzes the asymmetric hydrolysis of Ap4A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamide gel, a protein of 22 kDa exhibited Ap4A hydrolase activity. Both forms of the enzyme have a Km value in the range of 22 to 36 microM for Ap4A. Both forms of the enzyme asymmetrically hydrolyze Ap4A to AMP and ATP as determined by HPLC. Ap4A is the optimal substrate among several nucleotides and dinucleoside polyphosphates tested at 10 microM. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate Ap4A hydrolase while higher concentrations inhibit the activity. Pi is not a substrate for this Ap4A-degradative enzyme. Fluoride, from 50 microM to 20 mM, has no significant effect on Ap4A hydrolase activity.

Expression of organophosphorus-degradation gene ( opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

NASA Astrophysics Data System (ADS)

Li, Qiong; Tang, Qing; Xu, Xudong; Gao, Hong

2010-11-01

Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene ( opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase or human fecal cultures.

PubMed

Huijghebaert, S M; Hofmann, A F

1986-07-01

The influence of the chemical structure of the amino acid (or amino acid analogue) moiety of a number of synthetic cholyl amidates on deconjugation by cholylglycine hydrolase from Clostridium perfringens was studied in vitro at pH 5.4. Conjugates with alkyl homologues of glycine were hydrolyzed more slowly as the number of methylene units increased (cholylglycine greater than cholyl-beta-alanine greater than cholyl-gamma-aminobutyrate). In contrast, for conjugates with the alkyl homologues of taurine, cholylaminopropane sulfonate was hydrolyzed slightly faster than cholyltaurine, whereas cholylaminomethane sulfonate was hydrolyzed much more slowly. When glycine was replaced by other neutral alpha-amino acids, rates of hydrolysis decreased with increasing steric hindrance near the amide bond (cholyl-L-alpha-alanine much much greater than cholyl-L-leucine much greater than cholyl-L-valine greater than cholyl-L-tyrosine much greater than cholyl-D-valine). Conjugation with acidic or basic amino acids also greatly reduced the rates of hydrolysis, as cholyl-L-aspartate, cholyl-L-cysteate, cholyl-L-lysine, and cholyl-L-histidine were all hydrolyzed at a rate less than one-tenth that of cholylglycine. Methyl esterification of the carboxylic group of the amino acid moiety reduced the hydrolysis, but such substrates (cholylglycine methyl ester and cholyl-beta-alanine methyl ester) were completely hydrolyzed after overnight incubation with excess of enzyme. In contrast, cholyl-cholamine was not hydrolyzed at all, suggesting that a negative charge at the end of the side chain is required for optimal hydrolysis. Despite the lack of specificity for the amino acid moiety, a bile salt moiety was required, as the cholylglycine hydrolase did not display general carboxypeptidase activity for other non-bile acid substrates containing a terminal amide bond: hippuryl-L-phenylalanine and hippuryl-L-arginine, as well as oleyltaurine and oleylglycine, were not hydrolyzed. Fecal bacterial

Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay

EPA Science Inventory

Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay, Waalkes, M., Hunter, D.L., Jarema, K., Mundy, W., and S. Padilla. The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize organophosphor...

Environmental Fate of Organophosphorus Compounds Related to Chemical Weapons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davisson, M L; Love, A H; Vance, A

2005-02-08

Man-made organophosphorus compounds have been widely distributed throughout our environment as pesticides since their development during and after WWII. Many important studies have documented their relative persistence and toxicity. Development and use of some organophosphorus compounds as nerve agents gave rise to a separate but parallel effort to understand environmental persistence. In this latter case, the experiments have focused mainly on evaporation rates and first-order reaction kinetics. However, because organophosphorus compounds are easily polarized, the ionic content of a surrounding media directly factors into these reaction rates, but limited work in this regard has been done under environmentally relevant conditions.more » Furthermore, limited experiments investigating persistence of these agents on soil has resulted in widely varying degradation rates. Not surprisingly, no studies have investigated affinities of organophosphorus nerve agents to mineral or organic matter typically found in soil. As a result, we initiated laboratory experiments on dilute concentrations of nerve agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) to quantify persistence in simulated environmental aqueous conditions. A quantitative analytical method was developed for VX and its degradation products using High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS). VX hydrolysis rate is known to have a pH-dependency, however, the type of buffer and the relative proportion of different nucleophiles in solution significantly affect the overall rate and mechanism of degradation. For example, dissolved carbonate, a weak nucleophile dominating natural water, yielded pseudo-first order rate constants of {approx} 8 x 10{sup -3}/hr at pH 5 and 2 x 10{sup -2}/hr at pH 11. This small pH-dependent variation departs significantly from widely accepted rates at this pH range (4 x 10{sup -4}/hr to 8 x 10{sup -2}/hr) that were

Clinical emergency treatment of 68 critical patients with severe organophosphorus poisoning and prognosis analysis after rescue.

PubMed

Dong, Hui; Weng, Yi-Bing; Zhen, Gen-Shen; Li, Feng-Jie; Jin, Ai-Chun; Liu, Jie

2017-06-01

This study reports the clinical emergency treatment of 68 critical patients with severe organophosphorus poisoning, and analyzes the prognosis after rescue.The general data of 68 patients with severe organophosphorus poisoning treated in our hospital were retrospectively analyzed. These patients were divided into 2 groups: treatment group, and control group. Patients in the control group received routine emergency treatment, while patients in the treatment group additionally received hemoperfusion plus hemodialysis on the basis of routine emergency treatment. The curative effects in these 2 groups and the prognosis after rescue were compared.Compared with the control group, atropinization time, recovery time of cholinesterase activity, recovery time of consciousness, extubation time, and length of hospital stay were shorter (P < .05); the total usage of atropine was significantly lower (P < .05); Glasgow Coma Score was significantly higher (P < .05); acute physiology and chronic health score (APACHE II) was significantly lower (P < .05); and mortality and poisoning rebound rate was significantly lower (P < .05) in the treatment group.Hemoperfusion and hemodialysis on the basis of routine emergency treatment for critical patients with organophosphorus poisoning can improve rescue outcomes and improve the prognosis of patients, which should be popularized.

Sequence determination and analysis of S-adenosyl-L-homocysteine hydrolase from yellow lupine (Lupinus luteus).

PubMed

Brzeziński, K; Janowski, R; Podkowiński, J; Jaskólski, M

2001-01-01

The coding sequences of two S-adenosyl-L-homocysteine hydrolases (SAHases) were identified in yellow lupine by screenig of a cDNA library. One of them, corresponding to the complete protein, was sequenced and compared with 52 other SAHase sequences. Phylogenetic analysis of these proteins identified three groups of the enzymes. Group A comprises only bacterial sequences. Group B is subdivided into two subgroups, one of which (B1) is formed by animal sequences. Subgroup B2 consist of two distinct clusters, B2a and B2b. Cluster B2b comprises all known plant sequences, including the yellow lupine enzyme, which are distinguished by a 50-residue insert. Group C is heterogeneous and contains SAHases from Archaea as well as a new class of animal enzymes, distinctly different from those in group B1.

Engineering glycoside hydrolase stability by the introduction of zinc binding

DOE PAGES

Ellinghaus, Thomas L.; Pereira, Jose H.; McAndrew, Ryan P.; ...

2018-06-27

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activitymore » with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn 2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.« less

Engineering glycoside hydrolase stability by the introduction of zinc binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ellinghaus, Thomas L.; Pereira, Jose H.; McAndrew, Ryan P.

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activitymore » with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn 2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.« less

Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli.

PubMed

Radian, Adi; Aukema, Kelly G; Aksan, Alptekin; Wackett, Lawrence P

2015-11-03

waters, hypochlorite is frequently delivered as chlorinated isocyanuric acids that release hypochlorite and cyanuric acid. Over time, cyanuric acid accumulates and impairs disinfection and must be removed. The microbial enzyme cyanuric acid hydrolase can potentially remove cyanuric acid to restore disinfection and protect swimmers. Whole bacterial cells expressing cyanuric acid hydrolase were encapsulated in an inert silica matrix containing an amine group. The amine group serves to permeabilize the cell membrane and accelerate cyanuric acid degradation, and it also reacts with hypochlorite to protect against inactivation of cyanuric acid hydrolase. Methods for promoting whole-cell biocatalysis are important in biotechnology, and the present work illustrates approaches to enhance rates and protect against an inhibitory substance. Copyright © 2015 Radian et al.

Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

DOE PAGES

Chen, Mo; Bu, Lintao; Alahuhta, Markus; ...

2016-02-01

Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the productmore » inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.« less

Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Mo; Bu, Lintao; Alahuhta, Markus

Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the productmore » inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.« less

Effectiveness of organo-phosphorus insecticides against houseflies and mosquitos

PubMed Central

Lindquist, A. W.

1957-01-01

The paper describes the research being undertaken on organo-phosphorus insecticides for the control of houseflies and mosquitos. The information obtained from laboratory and field tests indicates that these insecticides are at present effective substitutes for DDT and other chlorinated-hydrocarbon insecticides for use against resistant houseflies and culicine mosquitos, but the residual applications are not as long lasting as those of DDT and therefore will probably not be as efficient in anopheline control. PMID:13413645

Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization

PubMed Central

1991-01-01

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and - deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6- P/IGF II receptor-deficient mouse J774 cells was more limited. beta- Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation

[Analysis on influencing factors of prognosis of patients with acute organophosphorus pesticide poisoning].

PubMed

Guo, C F; Wang, Y; Liu, J H; Shen, P; Wang, H; Wei, Y J; Shi, X F; Zhou, X J; Wang, W W

2016-05-20

To analyze the relationship between risk factors and prognosis of acute organophosphorus pesticide poisoning (AOPP). The clinical data including APACHEⅡ, D-dimer, C-reactive protein, procalcitonin, lactic acid of the 67 cases of acute organophosphorus pesticide poisoning which respectively divided into survival group and death group by the outcome were collected. The independent influcing factors were got by logistic regression analysis. The analysis showed that APACHEⅡ, D-dimer、C-reactive protein and Procalcitonin were influencing factors to evaluate prognosis of AOPP (P<0.05) .Meanwhile, APACHEⅡ and CRP were the independent influencing factors to evaluate prognosis of AOPP (P<0.05). APACHEⅡ26was the optimum thresholds to acess the prognosis of AOPP and its Youden index was largest. APACHEⅡ and CRP played an important role in the assessment of prognosis on AOPP. When APACHEⅡwas more than 26, it suggested the patient of AOPP will have a bad prognosis.

Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Brady D.; Apel, William A.; Sheridan, Peter P.

Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolasesmore » were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a

[Determination of 44 organophosphorus pesticides in food by SPE disk extraction-capillary gas chromatography with pulsed flame photometric detection].

PubMed

Luo, Xiao-Fei; Yang, Yuan; Sun, Cheng-Jun

2012-01-01

To develop a method for the simultaneous determination of 44 organophosphorus pesticides in food by SPE disk extraction-capillary gas chromatography with pulsed flame photometric detection. Organophosphorus pesticides in food were extracted ultrasonically with water. Then the extract was cleaned-up with SPE disk and eluted with ethyl acetate. Finally the eluent was condensed to 1mL under N2 at 55 degrees C. Gas chromatography was applied for quantitative detection of the organophosphorus pesticides in the sample. The linear range of the method for all the pesticides were in the range of 0.01-0.5 mg/kg with correlation coefficients of 0.992-1.000. The detection limits of the method were in the range of 0.0005-0.01 mg/kg. The recoveries for most pesticides were 60%-120% with relative standard deviations of less than 15%. The method is simple, sensitive, environmentally friendly and suitable for the determination of organophosphorous pesticides in food.

High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes

PubMed Central

Peat, Thomas S.; Balotra, Sahil; Wilding, Matthew; Hartley, Carol J.; Newman, Janet

2017-01-01

ABSTRACT The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity. IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically

Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents.

PubMed

Lushchekina, Sofya V; Schopfer, Lawrence M; Grigorenko, Bella L; Nemukhin, Alexander V; Varfolomeev, Sergei D; Lockridge, Oksana; Masson, Patrick

2018-01-01

Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants ( k cat / K m > 10 6 M -1 min -1 ) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing

Autolytic hydrolases affect sexual and asexual development of Aspergillus nidulans.

PubMed

Emri, Tamás; Vékony, Viktória; Gila, Barnabás; Nagy, Flóra; Forgács, Katalin; Pócsi, István

2018-03-30

Radial growth, asexual sporulation, and cleistothecia formation as well as extracellular chitinase and proteinase formation of Aspergillus nidulans were monitored in surface cultures in order to study the physiological role of extracellular hydrolase production in carbon-stressed cultures. We set up carbon-stressed and carbon-overfed experimental conditions by varying the starting glucose concentration within the range of 2.5 and 40 g/L. Glucose starvation induced radial growth and hydrolase production and enhanced the maturation of cleistothecia; meanwhile, glucose-rich conditions enhanced mycelial biomass, conidia, and cleistothecia production. Double deletion of chiB and engA (encoding an extracellular endochitinase and a β-1,3-endoglucanase, respectively) decreased conidia production under carbon-stressed conditions, suggesting that these autolytic hydrolases can support conidia formation by releasing nutrients from the cell wall polysaccharides of dead hyphae. Double deletion of prtA and pepJ (both genes encode extracellular proteases) reduced the number of cleistothecia even under carbon-rich conditions except in the presence of casamino acids, which supports the view that sexual development and amino acid metabolism are tightly connected to each other in this fungus.

Divergent effects of postmortem ambient temperature on organophosphorus- and carbamate-inhibited brain cholinesterase activity in birds

USGS Publications Warehouse

Hill, E.F.

1989-01-01

Time- and temperature-dependent postmortem changes in inhibited brain cholinesterase (ChE) activity may confound diagnosis of field poisoning of wildlife by anticholinesterase pesticide. Carbamate-inhibited ChE activity may return to normal within 1 to 2 days of exposure of intact carcass to moderate ambient temperature (18-32C). Organophosphorus-inhibited ChE activity becomes more depressed over the same time. Uninhibited ChE activity was resilient to above freezing temperature to 32C for 1 day and 25C for 3 days. Carbamate- and organophosphorus-inhibited ChE can be separated by incubation of homogenate for 1 hour at physiological temperatures; carbamylated ChE can be readily reactivated while phosphorylated ChE cannot.

Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins

PubMed Central

De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2017-01-01

The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination. PMID:23035660

Structure-Guided Engineering of Molinate Hydrolase for the Degradation of Thiocarbamate Pesticides

PubMed Central

Paiva, Ana M.; Ferreira-da-Silva, Frederico; Matias, Pedro M.; Nunes, Olga C.; Gales, Luís

2015-01-01

Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system. PMID:25905461

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

PubMed Central

Knudsen, J

1979-01-01

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue. PMID:574008

Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

PubMed

Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

2016-01-01

Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences. Copyright © 2015 Elsevier B.V. All rights reserved.

Osmoregulatory function in ducks following ingestion of the organophosphorus insecticide fenthion

USGS Publications Warehouse

Rattner, B.A.; Fleming, W.J.; Murray, H.C.

1983-01-01

Salt gland function and osmoregulation in aquatic birds drinking hyperosmotic water has been suggested to be impaired by organophosphorus insecticides. To test this hypothesis, adult black ducks (Anas rubripes) were provided various regimens of fresh or salt (1.5% NaCl) water before, during, and after ingestion of mash containing 21 ppm fenthion. Ducks were bled by jugular venipuncture after I, 7. and 12 days of treatment, and were then killed. Brain and salt gland acetylcholinesterase activities were substantially inhibited (44-61% and 14-36%) by fenthion. However, salt gland weight and Na + -K + -ATPase activity, and plasma Na + , CI- , and osmolality, were uniformly elevated in all groups receiving salt water including those ingesting fenthion. In a second study, salt gland Na + -K + -ATPase activity in mallards (A. platyrhynchos) was not affected after in vitro incubation with either fenthion or fenthion oxon at concentrations ranging from 0.04 to 400 ?M, but was reduced in the presence of 40 and 400 ?M DDE (positive control). These findings suggest that environmentally realistic concentrations of organophosphorus insecticides do not markedly affect osmoregulatory function in adult black ducks.

Possible Correlation Between Bile Salt Hydrolysis and AHL Deamidation: Staphylococcus epidermidis RM1, a Potent Quorum Quencher and Bile Salt Hydrolase Producer.

PubMed

Mukherji, Ruchira; Prabhune, Asmita

2015-05-01

The aim of the present work was to isolate a bile salt hydrolase (BSH) producer from fermented soy curd and explore the ability of the BSH produced to cleave bacterial quorum sensing signals. Bacterial isolates with possible ability to deconjugate bile salts were enriched and isolated on De Man, Rogosa and Sharpe (MRS) medium containing 0.2% bile salts. BSH-producing positive isolate with orange-pink-pigmented colonies was isolated and was identified as a strain of Staphylococcus epidermidis using biochemical and phylogenetic tools. S. epidermidis RM1 was shown to possess both potent BSH and N-acyl homoserine lactone (AHL) cleavage activity. Genetic basis of this dual-enzyme activity was explored by means of specific primers designed using S. epidermidis ATCC 12228 genome as template. It was observed that a single enzyme was not responsible for both the activity. Two different genetic elements corresponding to each of the enzymatic activity were successfully amplified from the genomic DNA of the isolate.

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α-l-Rhamnosidases from Pediococcus acidilactici▿†

PubMed Central

Michlmayr, Herbert; Brandes, Walter; Eder, Reinhard; Schümann, Christina; del Hierro, Andrés M.; Kulbe, Klaus D.

2011-01-01

α-l-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ram and ram2) from Pediococcus acidilactici DSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial β-glucosidase, both enzymes released the monoterpenes linalool and cis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substrate p-nitrophenyl-α-l-rhamnopyranoside (Vmax = 243 U mg−1). In contrast, Ram2 displayed considerable specificity toward hesperidin (Vmax = 34 U mg−1) and, especially, rutinose (Vmax = 1,200 U mg−1), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases. PMID:21784921

Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design.

PubMed

Melzer, Marco; Chen, Julian C-H; Heidenreich, Anne; Gäb, Jürgen; Koller, Marianne; Kehe, Kai; Blum, Marc-Michael

2009-12-02

Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.

Biodegradation of brominated and organophosphorus flame retardants.

PubMed

Waaijers, Susanne L; Parsons, John R

2016-04-01

Brominated flame retardants account for about 21% of the total production of flame retardants and many of these have been identified as persistent, bioaccumulative and toxic. Nevertheless, debromination of these chemicals under anaerobic conditions is well established, although this can increase their toxicity. Consequently, the production and use of these chemicals has been restricted and alternative products have been developed. Many of these are brominated compounds and share some of the disadvantages of the chemicals they are meant to replace. Therefore, other, nonbrominated, flame retardants such as organophosphorus compounds are also being used in increasing quantities, despite the fact that knowledge of their biodegradation and environmental fate is often lacking. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundeen, S.G.; Savage, D.C.

1990-08-01

The authors have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving ({sup 14}C)taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in mediummore » free of bile salts. In cell-free extracts, however, the activity was about equal whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Whether the enzyme exists in two forms in the cells remains to be determined.« less

Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

Consolidation of glycosyl hydrolase family 30 : a dual domain 4/7 hydrolase family consisting of two structurally distinct groups

Treesearch

Franz J. St John; Javier M. Gonzalez; Edwin Pozharski

2010-01-01

In this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement...

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

PubMed

Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

2016-08-01

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

DEVELOPMENT OF RISK ASSESSMENT TECHNIQUES FOR SOIL BIOAVAILABILITY AND BIOACCUMULATION OF AN ORGANOPHOSPHORUS PESTICIDE

EPA Science Inventory

There is increased concern about the effects of organophosphorus (OP) pesticides on human and animal health. This class of chemicals has been shown to affect the immune function of macrophages and lymphocytes. Malathion, an OP compound, is one of the most widely used pesticides...

Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

USDA-ARS?s Scientific Manuscript database

The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

Enzymatic degradation of monocrotophos by extracellular fungal OP hydrolases.

PubMed

Jain, Rachna; Garg, Veena

2013-11-01

The present study explores the potential of extracellular fungal organophosphate (OP) hydrolase for the degradation of monocrotophos. Extracellular OP hydrolases were isolated and purified from five different fungal isolates viz. Aspergillus niger (M1), Aspergillus flavus (M2), Penicillium aculeatum (M3), Fusarium pallidoroseum (M4), and Macrophomina sp. (M5) by AmSO4 precipitation, dialysis, and G-100 chromatography. M3 showed highest percentage yield of 68.81 followed by 55.41 % for M1. Each of the purified enzyme fraction constituted of two different subunits of 33- and 67-kDa molecular weight. Optimum enzyme fraction (150 μg ml(-1)) rapidly degraded monocrotophos within 120 h in phosphorus-free liquid culture medium (CZM) with K deg of 0.0368, 0.0138, 0.048, 0.016, 0.0138, and 0.048 day(-1) and half-life of 0.79, 2.11, 0.6, 1.8, and 2.11 days for M1, M2, M3, M4, and M5, respectively. The results were further confirmed by high performance thin layer chromatography and Fourier transform infrared which indicate the disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. The overall order of enzymatic degradation was found to be P. aculeatum > A. niger > F. pallidoroseum > A. flavus = Macrophomina sp. Hence, the study concludes that extracellular OP hydrolases efficiently degraded monocrotophos and could be used as a potential candidate for the detoxification of this neurotoxin pesticide.

Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

DTIC Science & Technology

1985-07-01

transient increase in the salicylic acid hydrolysis product was observed. Pretreatment with 4-nitrophenyl methyl(phenyl)phosphinate had no significant...h. Hydroly- sis of aspirin was not reduced in pretreated mice, although a transient increase in the salicylic acid hydrolysis product was observed...26 Figure 1. Pathways of aspirin metabolism in mammals: CE is carboxylester hydrolase, SA is salicylic acid, SU is salicyluric

Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

DOE Office of Scientific and Technical Information (OSTI.GOV)

Germane, Katherine L., E-mail: katherine.germane.civ@mail.mil; Servinsky, Matthew D.; Gerlach, Elliot S.

2015-07-29

The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes themore » unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

MOVEMENT AND DEPOSITION OF TWO ORGANOPHOSPHORUS PESTICIDES WITHIN A RESIDENCE AFTER INTERIOR AND EXTERIOR APPLICATIONS

EPA Science Inventory

Post-application temporal and spatial distributions of two organophosphorus pesticides, diazinon and chlorpyrifos, were monitored after homeowner applications for indoor and outdoor insect control. Samples were taken before and up to 12 days after treatments in the family room...

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth.

PubMed

Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M L; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

2016-01-01

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro . We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia . By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

PubMed Central

Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M. L.; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

2016-01-01

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present. PMID:27729900

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF THE ENANTIOMERS OF ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

EPA Science Inventory

High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) was obtained on polysaccharide enantioselective HPLC columns using alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, fonofos, fenamiph...

EFFECTS OF TEMPERATURE AND SOLVENT COMPOSITION ON THE CHIRALCEL OJ SEPARATION OF CHIRAL ORGANOPHOSPHORUS PESTICIDES

EPA Science Inventory

The separation of the enantiomers of twelve organophosphorus pesticides (OPs) was investigated on the CHIRALCEL?OJ column to determine whether the mobile phase composition, flow rate and column temperature could be optimized to yield at least partial separation of the enantiomers...

Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a.

PubMed

Hu, Wenxin; Wang, Qihai; Bi, Ruchang

2005-12-01

Diadenosine tetraphosphate (Ap4A) hydrolase (EC 3.6.1.41) hydrolyzes Ap4A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap4A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap4A hydrolase crystals diffract X-rays to 3.26 A and belong to space group P2(1), with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 A, beta = 95.7 degrees.

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

PubMed

Mangas, I; Vilanova, E; Benabent, M; Estévez, J

2014-02-10

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Evaluation of Chemically-Sensitive Field-Effect Transistors for Detection of Organophosphorus Compounds

DTIC Science & Technology

1989-12-05

during past decade. In order to understand the basic operation of these sensors, especially of the CHEMFET, the appropriate background information will...during the past decade for detecting organophosphorus compounds, the chemically- sensitive thin films investigated in this thesis, and finally, the...reactivate the phosphorylated cholinesterase enzyme. Solid State Chemical Sensors During the past decade, a number of solid state chemical sensors have been

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis1[W

PubMed Central

Ogawa, Takahisa; Yoshimura, Kazuya; Miyake, Hiroe; Ishikawa, Kazuya; Ito, Daisuke; Tanabe, Noriaki; Shigeoka, Shigeru

2008-01-01

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its Km value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 μm) than for ADP-glucose (1,235 ± 65 μm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 μm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 μm) and NADPH (36.9 ± 3.5 μm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 μm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes. PMID:18815383

Recognition of corn defense chitinases by fungal polyglycine hydrolases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific pe...

Effects of Organophosphorus Flame Retardants on Spontaneous Activity in Neuronal Networks Grown on Microelectrode Arrays

EPA Science Inventory

EFFECTS OF ORGANOPHOSPHORUS FLAME RETARDANTS ON SPONTANEOUS ACTIVITY IN NEURONAL NETWORKS GROWN ON MICROELECTRODE ARRAYS TJ Shafer1, K Wallace1, WR Mundy1, M Behl2,. 1Integrated Systems Toxicology Division, NHEERL, USEPA, RTP, NC, USA, 2National Toxicology Program, NIEHS, RTP, NC...

Furthering the Enzymatic Destruction of Nerve Agents

DTIC Science & Technology

2002-01-01

properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate. J. Bacteriol. 173, 1938-1943. DeFrank, J.J.; Beaudry, W.T...Cheng, T.-c.; Harvey, S.P.; Stroup, A.N. and Szafraniec, L.L. (1993) Screening of halophilic bacteria and Alteromonas species for organophosphorus

Soda pans of the Pannonian steppe harbor unique bacterial communities adapted to multiple extreme conditions.

PubMed

Szabó, Attila; Korponai, Kristóf; Kerepesi, Csaba; Somogyi, Boglárka; Vörös, Lajos; Bartha, Dániel; Márialigeti, Károly; Felföldi, Tamás

2017-05-01

Soda pans of the Pannonian steppe are unique environments regarding their physical and chemical characteristics: shallowness, high turbidity, intermittent character, alkaline pH, polyhumic organic carbon concentration, hypertrophic condition, moderately high salinity, sodium and carbonate ion dominance. The pans are highly productive environments with picophytoplankton predominance. Little is known about the planktonic bacterial communities inhabiting these aquatic habitats; therefore, amplicon sequencing and shotgun metagenomics were applied to reveal their composition and functional properties. Results showed a taxonomically complex bacterial community which was distinct from other soda lakes regarding its composition, e.g. the dominance of class Alphaproteobacteria was observed within phylum Proteobacteria. The shotgun metagenomic analysis revealed several functional gene components related to the harsh and at the same time hypertrophic environmental conditions, e.g. proteins involved in stress response, transport and hydrolase systems targeting phytoplankton-derived organic matter. This is the first detailed report on the indigenous planktonic bacterial communities coping with the multiple extreme conditions present in the unique soda pans of the Pannonian steppe.

Isolation and characterization of bacteria capable of degrading polycyclic aromatic hydrocarbons (PAHs) and organophosphorus pesticides from PAH-contaminated soil in Hilo, Hawaii.

PubMed

Seo, Jong-Su; Keum, Young-Soo; Harada, Renee M; Li, Qing X

2007-07-11

Nineteen bacterial strains were isolated from petroleum-contaminated soil in Hilo, HI, and characterized by two different spray-plated methods, turbidity test in liquid medium, and 16S rRNA gene sequence analysis. Analysis of the soil showed 13 polycyclic aromatic hydrocarbons (PAHs) in a range from 0.6 to 30 mg/kg of dry weight each and 12 PAH metabolites. Five distinct bacterial strains (C3, C4, P1-1, JS14, and JS19b1) selected from preliminary plating and turbidity tests were further tested for PAH degradation through single PAH degradation assay. Strains C3, C4, and P1-1 degraded phenanthrene (40 mg/L) completely during 7 days of incubation. Strain JS14 degraded fluoranthene (40 mg/L) completely during 10 days of incubation. Strain JS19b1 degraded 100% of phenanthrene (40 mg/L) in 7 days, 77% of fluorene (40 mg/L) in 14 days, 97% of fluoranthene (40 mg/L) in 10 days, and 100% of pyrene (40 mg/L) in 14 days. Turbidity tests showed that strains P1-1, JS14, and JS19b1 utilized several organophosphorus pesticides as growth substrate. P1-1 can degrade carbofenothion, chlorfenvinphos, diazinon, fonofos, and pirimiphos-methyl. JS14 can transform chlorfenvinphos and diazinon. JS19b1 can break down diazinon, pirimiphos-methyl, and temephos.

COMPARISON OF THE NON-ADDITIVE INTERACTIONS OF AN ORGANOPHOSPHORUS PESTICIDE MIXTURE IN ADULT AND PREWEANLING RATS.

EPA Science Inventory

Critical features of risk assessment include the evaluation of risk following exposure to pesticide mixtures as well as the potential for increased sensitivity of the young. This research tested for interaction(s) using a mixture of five organophosphorus (OP) pesticides (chlorp...

Use of Alternative Assays to Identify and Prioritize Organophosphorus Flame Retardants for Potential Developmental and Neurotoxicity

EPA Science Inventory

Due to their toxicity and persistence in the environment, brominated flame retardants (BFRs) are being phased out of commercial use, leading to the increased use of alternative chemicals such as the organophosphorus flame retardants (OPFRs). There is, however, limited information...

Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

PubMed Central

Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

2015-01-01

The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications. PMID:25854643

Structural genomics analysis of uncharacterized protein families overrepresented in human gut bacteria identifies a novel glycoside hydrolase

PubMed Central

2014-01-01

Background Bacteroides spp. form a significant part of our gut microbiome and are well known for optimized metabolism of diverse polysaccharides. Initial analysis of the archetypal Bacteroides thetaiotaomicron genome identified 172 glycosyl hydrolases and a large number of uncharacterized proteins associated with polysaccharide metabolism. Results BT_1012 from Bacteroides thetaiotaomicron VPI-5482 is a protein of unknown function and a member of a large protein family consisting entirely of uncharacterized proteins. Initial sequence analysis predicted that this protein has two domains, one on the N- and one on the C-terminal. A PSI-BLAST search found over 150 full length and over 90 half size homologs consisting only of the N-terminal domain. The experimentally determined three-dimensional structure of the BT_1012 protein confirms its two-domain architecture and structural analysis of both domains suggests their specific functions. The N-terminal domain is a putative catalytic domain with significant similarity to known glycoside hydrolases, the C-terminal domain has a beta-sandwich fold typically found in C-terminal domains of other glycosyl hydrolases, however these domains are typically involved in substrate binding. We describe the structure of the BT_1012 protein and discuss its sequence-structure relationship and their possible functional implications. Conclusions Structural and sequence analyses of the BT_1012 protein identifies it as a glycosyl hydrolase, expanding an already impressive catalog of enzymes involved in polysaccharide metabolism in Bacteroides spp. Based on this we have renamed the Pfam families representing the two domains found in the BT_1012 protein, PF13204 and PF12904, as putative glycoside hydrolase and glycoside hydrolase-associated C-terminal domain respectively. PMID:24742328

[On necessity to modify biochemical methods for detecting organophosphorus componds in chemical weapons extinction objects (review of literature)].

PubMed

Prokofieva, D S; Shmurak, V I; Sadovnikov, S V; Gontcharov, N V

2015-01-01

The article covers problems of biochemical methods assessing organophosphorus toxic compounds in objects of chemical weapons extinction. The authors present results of works developing new, more specific and selective biochemical methods.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

PubMed

Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

2016-02-01

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transformation of Organophosphorus Pesticides in the Presence of Aqueous Chlorine: Kinetics, Pathways, and Structure-Activity Relationships

EPA Science Inventory

The fate of organophosphorus (OP) pesticides in the presence of aqueous chlorine was investigated under simulated drinking water treatment conditions. Intrinsic rate coefficients were found for the reaction of hypochlorous acid (kHOCl,OP) and hypochlorite ion (kOCl,OP) for eight...

Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

PubMed

Wong, Fong T; Hotta, Kinya; Chen, Xi; Fang, Minyi; Watanabe, Kenji; Kim, Chu-Young

2015-01-14

Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme

PubMed Central

Schoefer, Lilian; Braune, Annett; Blaut, Michael

2004-01-01

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively. The Km for phloretin was 13 ± 3 μM and the kcat was 10 ± 2 s−1. The enzyme did not transform phloretin-2′-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl2 to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively. PMID:15466559

Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pecic, Stevan; Pakhomova, Svetlana; Newcomer, Marcia E.

2013-09-27

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.

Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quistad, Gary B.; Klintenberg, Rebecka; Caboni, Pierluigi

2006-02-15

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicalsmore » (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C{sub 1}, C{sub 2}, C{sub 3}) alkylphosphonofluoridates (C{sub 8}, C{sub 12}) (IC50 0.60-3.0 nM), five S-alkyl (C{sub 5}, C{sub 7}, C{sub 9}) and alkyl (C{sub 1}, C{sub 12}) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.« less

[Decontamination of organophosphorus compounds: Towards new alternatives].

PubMed

Poirier, L; Jacquet, P; Elias, M; Daudé, D; Chabrière, E

2017-05-01

Organophosphorus coumpounds (OP) are toxic chemicals mainly used for agricultural purpose such as insecticides and were also developed and used as warfare nerve agents. OP are inhibitors of acetylcholinesterase, a key enzyme involved in the regulation of the central nervous system. Chemical, physical and biological approaches have been considered to decontaminate OP. This review summarizes the current and emerging strategies that are investigated to tackle this issue with a special emphasis on enzymatic remediation methods. During the last decade, many studies have been dedicated to the development of biocatalysts for OP removal. Among these, recent reports have pointed out the promising enzyme SsoPox isolated from the archaea Sulfolobus solfataricus. Considering both its intrinsic stability and activity, this hyperthermostable enzyme is highly appealing for the decontamination of OP. Copyright © 2017 Académie Nationale de Pharmacie. All rights reserved.

Comprehensive cross-genome survey and phylogeny of Glycoside Hydrolase Family 16 members reveals the evolutionary origin of EG16 and XTH proteins in plant lineages.

PubMed

Behar, Hila; Graham, Sean W; Brumer, Harry

2018-06-22

Carbohydrate-active enzymes (CAZymes) are central to the biosynthesis and modification of the plant cell wall. An ancient clade of bifunctional plant endo-glucanases (EG16 members) was recently revealed and proposed to represent a transitional group uniting plant xyloglucan endo-transglycosylase/hydrolase (XTH) gene products and bacterial mixed-linkage endo-glucanases in the phylogeny of Glycoside Hydrolase Family 16 (GH16). To gain broader insights into the distribution and frequency of EG16 and other GH16 members in plants, the Phytozome, Plaza, NCBI, and 1000 Plants databases were mined to build a comprehensive census among 1289 species spanning the broad phylogenetic diversity of multiple algae through recent plant lineages. EG16, newly identified EG16-2, and XTH members appeared first in the green algae. Extant EG16 members represent the early adoption of the β-jelly-roll protein scaffold from a bacterial or early-lineage eukaryotic GH16 gene, which is characterized by loop deletion and extension of the N-terminus (in EG16-2 members) or C-terminus (in XTH members). Maximum-likelihood phylogenetic analysis of EG16 and EG16-2 sequences are directly concordant with contemporary estimates of plant evolution. The lack of expansion of EG16 members into multi-gene families across green plants may point to a core metabolic role under tight control, in contrast to XTH genes that have undergone extensive duplications typical of cell-wall CAZymes. The present census will underpin future studies to elucidate the physiological role of EG16 members across plant species, and serve as roadmap for delineating the closely related EG16 and XTH gene products in bioinformatic analyses of emerging genomes and transcriptomes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

[Effects of nitrogen additions on soil hydrolase and oxidase activities in Pinus elliottii plantations.

PubMed

Zhang, Chuang; Zou, Hong Tao; Zhang, Xin Yu; Kou, Liang; Yang, Yang; Sun, Xiao Min; Li, Sheng Gong; Wang, Hui Min

2016-11-18

We evaluated responses of hydrolase and oxidase activities in a subtropical Pinus elliottii plantation through a nitrogen (N) addition field experiment (dosage level: 0, 40, 120 kg N·hm -2 ·a -1 ). The results showed that N additions significantly decreased the carbon, nitrogen and phosphorus related hydrolase and oxidase activities. The activities of β-1,4-glucosidase (BG), cellobiohydrolase (CBH), β-1,4-N-acetylglucosaminidase (NAG) and peroxidase (PER) activities were decreased by 16.5%-51.1% due to N additions, and the decrease was more remarkable in the higher N addition treatment. The activities of α-1,4-glucosidase (aG), β-1,4-xylosidase (BX), acid phosphatase (AP) and phenol oxidase (PPO) were decreased by 14.5%-38.6% by N additions, however, there was no significant difference among the different N addition treatments. Soil enzyme activities varied obviously in different seasons. The activities of BG, NAG, BX, CBH, AP and PPO were in the order of March > June > October, and aG and PER activities were in the order of October > March > June. Most of the soil hydrolase and oxidase activities were positively correlated with soil pH, but negatively with NO 3 - -N content. It indicated that N additions inhibited soil hydrolase and oxidase activities by reducing soil pH and increasing soil nitrification. N additions inhibited the soil organic matter mineralization and turnover in the subtropical area, and the effects were obvious with the increasing dosage of N additions.

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family*

PubMed Central

De Jaco, Antonella; Lin, Michael Z.; Dubi, Noga; Comoletti, Davide; Miller, Meghan T.; Camp, Shelley; Ellisman, Mark; Butko, Margaret T.; Tsien, Roger Y.; Taylor, Palmer

2010-01-01

Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems. PMID:20615874

Neuroligin trafficking deficiencies arising from mutations in the alpha/beta-hydrolase fold protein family.

PubMed

De Jaco, Antonella; Lin, Michael Z; Dubi, Noga; Comoletti, Davide; Miller, Meghan T; Camp, Shelley; Ellisman, Mark; Butko, Margaret T; Tsien, Roger Y; Taylor, Palmer

2010-09-10

Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.

Structural insight into catalytic mechanism of PET hydrolase.

PubMed

Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

2017-12-13

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

PubMed

Stahl, U.; Banas, A.; Stymne, S.

1995-03-01

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

Repurposing Suzuki Coupling Reagents as a Directed Fragment Library Targeting Serine Hydrolases and Related Enzymes.

PubMed

Lanier, Marion; Cole, Derek C; Istratiy, Yelena; Klein, Michael G; Schwartz, Phillip A; Tjhen, Richard; Jennings, Andy; Hixon, Mark S

2017-06-22

Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease.

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bera, Asim K.; Aukema, Kelly G.; Elias, Mikael

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas themore » two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.« less

Regio- and enantiofacial selectivity of epoxyeicosatrienoic acid hydration by cytosolic epoxide hydrolase.

PubMed

Zeldin, D C; Kobayashi, J; Falck, J R; Winder, B S; Hammock, B D; Snapper, J R; Capdevila, J H

1993-03-25

The hydration of cis-epoxyeicosatrienoic acids to the corresponding vic-dihydroxyeicosatrienoic acids by cytosolic epoxide hydrolase demonstrates moderate regioselectivity with rates of hydration highest for the 14,15-epoxide and lower for the 11,12- and 8,9-epoxide (4.5, 1.6, and 1.5 mumol of product/mg of protein/min, respectively). Incubations of the 8,9- and 14,15-epoxides with cytosolic epoxide hydrolase show stereoselective formation of diols (7:3 and 4:1 ratio of antipodes, respectively) and concomitant chiral enrichment of the remaining unmetabolized substrate. In contrast, hydration of the 11,12-epoxide is nonenantioselective. The Km value of the enzyme for the 14(R),15(S)-epoxide is 3 microM. Incubations of the enantiomerically pure 8,9- and 14,15-epoxides with lung or liver cytosol, followed by chiral analysis of the resulting diols demonstrate selective cleavage of the oxirane ring at C9 and C15, respectively. On the other hand, cleavage of the 11,12- oxirane ring was less selective. The stereochemical preference of the cytosolic epoxide hydrolase, together with the known chiral composition of the endogenous arachidonate epoxide pools, suggests a functional role for this enzyme in the metabolism of these important compounds.

Novel choline esterase based sensor for monitoring of organophosphorus pollutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkins, E.S.; Ghindilis, A.L.; Atanasov, P.

1996-12-31

Organophosphorus compounds are significant major environmental pollutants due to their intensive use as pesticides. The modern techniques based on inhibition of choline esterase enzyme activity are discussed. Potentiometric electrodes based on detection of choline esterase inhibition by analytes has been developed. The detection of choline esterase activity is based on the novel principle of molecular transduction. Immobilized peroxidase acting as the molecular transducer, catalyzes the electroreduction of hydrogen peroxide by direct (mediatorless) electron transfer. The sensing element consists of a carbon based electrode containing an assembly of co-immobilized enzymes: choline esterase, choline oxidase and peroxidase.

Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

John, George; Nagarajan, Subbiah; Chapman, Kent

A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacyl-ethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

PubMed

Oguro, Ami; Imaoka, Susumu

2012-03-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

PubMed

Miyazaki, Takatsugu; Ishizaki, Yuichi; Ichikawa, Megumi; Nishikawa, Atsushi; Tonozuka, Takashi

2015-07-01

Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family. © 2015 Authors; published by Portland Press Limited.

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

PubMed

Christiaens, H; Leer, R J; Pouwels, P H; Verstraete, W

1992-12-01

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

Saccharification of woody biomass using glycoside hydrolases from Stereum hirsutum.

PubMed

Jeya, Marimuthu; Kalyani, Dayanand; Dhiman, Saurabh Sudha; Kim, Hoon; Woo, Seongmin; Kim, Dongwook; Lee, Jung-Kul

2012-08-01

Enzymatic saccharification of woody biomasses was performed using glycoside hydrolases from Stereum hirsutum, a newly isolated fungal strain found to secrete efficient glycoside hydrolases. The strain showed the highest β-glucosidase, cellobiohydrolase, endoglucanase, endoxylanase, laccase, and filter paper activity of 10.3, 1.7, 10.3, 29.9, 0.12, and 0.58 U/ml, respectively. Among the various biomasses tested for saccharification, pine biomass produced maximum reducing sugar. Response surface methodology was used to optimize the hydrolysis of pine biomass to achieve the highest level of sugars. The parameters including enzyme, substrate concentration, temperature and pH were found to be critical for the conversion of pine biomass into sugars. Maximum saccharification of 49.7% (435 mg/g-substrate) was obtained after 96 h of hydrolysis. A close agreement between the experimental results and the model predictions was achieved. S. hirsutum could be a good choice for the production of reducing sugars from cellulosic biomasses. Copyright © 2012 Elsevier Ltd. All rights reserved.

Assessing in silico the recruitment and functional spectrum of bacterial enzymes from secondary metabolism.

PubMed

Veprinskiy, Valery; Heizinger, Leonhard; Plach, Maximilian G; Merkl, Rainer

2017-01-26

Microbes, plants, and fungi synthesize an enormous number of metabolites exhibiting rich chemical diversity. For a high-level classification, metabolism is subdivided into primary (PM) and secondary (SM) metabolism. SM products are often not essential for survival of the organism and it is generally assumed that SM enzymes stem from PM homologs. We wanted to assess evolutionary relationships and function of bona fide bacterial PM and SM enzymes. Thus, we analyzed the content of 1010 biosynthetic gene clusters (BGCs) from the MIBiG dataset; the encoded bacterial enzymes served as representatives of SM. The content of 15 bacterial genomes known not to harbor BGCs served as a representation of PM. Enzymes were categorized on their EC number and for these enzyme functions, frequencies were determined. The comparison of PM/SM frequencies indicates a certain preference for hydrolases (EC class 3) and ligases (EC class 6) in PM and of oxidoreductases (EC class 1) and lyases (EC class 4) in SM. Based on BLAST searches, we determined pairs of PM/SM homologs and their functional diversity. Oxidoreductases, transferases (EC class 2), lyases and isomerases (EC class 5) form a tightly interlinked network indicating that many protein folds can accommodate different functions in PM and SM. In contrast, the functional diversity of hydrolases and especially ligases is significantly limited in PM and SM. For the most direct comparison of PM/SM homologs, we restricted for each BGC the search to the content of the genome it comes from. For each homologous hit, the contribution of the genomic neighborhood to metabolic pathways was summarized in BGC-specific html-pages that are interlinked with KEGG; this dataset can be downloaded from https://www.bioinf.ur.de . Only few reaction chemistries are overrepresented in bacterial SM and at least 55% of the enzymatic functions present in BGCs possess PM homologs. Many SM enzymes arose in PM and Nature utilized the evolvability of enzymes

Comparison of the Developmental and Acute Neurotoxicity of a Library of Organophosphorus Pesticides Using a Vertebrate Behavioral Assay

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize organophosphorus pesticides for neurotoxicity using behavioral tests in an in vivo, vertebrate, medium-throughput model (zebrafish; Danio rerio). Our behavioral testing paradigm assesses the e...

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

PubMed Central

Stahl, U.; Banas, A.; Stymne, S.

1995-01-01

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

Exploring the bacterial gut microbiota of supralittoral talitrid amphipods.

PubMed

Abdelrhman, Khaled F A; Bacci, Giovanni; Marras, Barbara; Nistri, Annamaria; Schintu, Marco; Ugolini, Alberto; Mengoni, Alessio

2017-01-01

Talitrid amphipods (sandhoppers and beach fleas) are typical of the supralittoral zone. They are known to thrive on stranded materials, including detrital marine angiosperms and macroalgae, as well as occasional dead animals. In this work, the gut microbiota of five species of talitrid amphipods (Talitrus saltator, Talorchestia ugolinii, Sardorchestia pelecaniformis, Orchestia montagui and Orchestia stephenseni) collected in Sardinia (Italy) has been investigated through: i) metabarcoding analysis of the amplified 16S rRNA V4 region; and ii) quantification of family 48 glycosyl hydrolase genes (GHF48), involved in cellulose degradation. Results indicate that, though talitrid gut biodiversity is not directly related to taxon or sampling locality, the animals' digestive tracts may host species-specific bacterial communities. In particular, gut microbiota of O. montagui, an inhabitant of Posidonia banquettes and macro-algae mat, showed the greatest differences in taxonomic composition and the highest proportion of GHF48 genes with respect to 16S rRNA genes. These results suggest that the different talitrid species may host species-specific bacterial communities whose function could partially reflect the different microhabitats and food preferences of their host. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Recent Developments in Organophosphorus Flame Retardants Containing P-C Bond and Their Applications

PubMed Central

Wendels, Sophie; Chavez, Thiebault; Bonnet, Martin; Gaan, Sabyasachi

2017-01-01

Organophosphorus compounds containing P-C bonds are increasingly developed as flame retardant additives due to their excellent thermal and hydrolytic stability and ease of synthesis. The latest development (since 2010) in organophosphorus flame retardants containing P-C bonds summarized in this review. In this review, we have broadly classified such phosphorus compounds based on the carbon unit linked to the phosphorus atom i.e., could be a part of either an aliphatic or an aromatic unit. We have only considered those published literature where a P-C bond was created as a part of synthetic strategy to make either an intermediate or a final organophosphorus compound with an aim to use it as a flame retardant. General synthetic strategies to create P-C bonds are briefly discussed. Most popular synthetic strategies used for developing P-C containing phosphorus based flame retardants include Michael addition, Michaelis–Arbuzov, Friedels–Crafts and Grignard reactions. In general, most flame retardant derivatives discussed in this review have been prepared via a one- to two-step synthetic strategy with relatively high yields greater than 80%. Specific examples of P-C containing flame retardants synthesized via suitable synthetic strategy and their applications on various polymer systems are described in detail. Aliphatic phosphorus compounds being liquids or low melting solids are generally applied in polymers via coatings (cellulose) or are incorporated in the bulk of the polymers (epoxy, polyurethanes) during their polymerization as reactive or non-reactive additives. Substituents on the P atoms and the chemistry of the polymer matrix greatly influence the flame retardant behavior of these compounds (condensed phase vs. the gas phase). Recently, aromatic DOPO based phosphinate flame retardants have been developed with relatively higher thermal stabilities (>250 °C). Such compounds have potential as flame retardants for high temperature processable polymers such as

A Common Mechanism for Resistance to Oxime Reactivation of Acetylcholinesterase Inhibited by Organophosphorus Compounds

DTIC Science & Technology

2013-01-01

application of the Hammett equation with the constants rph in the chemistry of organophosphorus compounds, Russ. Chem. Rev. 38 (1969) 795–811. [13...of oximes and OP compounds and the ability of oximes to reactivate OP- inhibited AChE. Multiple linear regression equations were analyzed using...phosphonate pairs, 21 oxime/ phosphoramidate pairs and 12 oxime/phosphate pairs. The best linear regression equation resulting from multiple regression anal

Organophosphorus and Organochlorine Pesticides Bioaccumulation by Eichhornia crassipes in Irrigation Canals in an Urban Agricultural System.

PubMed

Mercado-Borrayo, B M; Cram Heydrich, Silke; Pérez, Irma Rosas; Hernández Quiroz, Manuel; De León Hill, Claudia Ponce

2015-01-01

A natural wetland in Mexico City Metropolitan Area is one of the main suppliers of crops and flowers, and in consequence its canals hold a high concentration of organochlorine (OC) and organophosphorus (OP) pesticides. There is also an extensive population of water hyacinth (Eichhornia crassipes), which is considered a plague; but literature suggests water hyacinth may be used as a phytoremediator. This study demonstrates bioaccumulation difference for the OC in vivo suggesting their bioaccumulation is ruled by their log K(ow), while all the OP showed bioaccumulation regardless of their log K(ow). The higher bioaccumulation factors (BAF) of the accumulated OC pesticides cannot be explained by their log K(ow), suggesting that the OC pesticides may also be transported passively into the plant. Translocation ratios showed that water hyacinth is an accumulating plant with phytoremediation potential for all organophosphorus pesticides studied and some organochlorine pesticides. An equation for free water surface wetlands with floating macrophytes, commonly used for the construction of water-cleaning wetlands, showed removal of the pesticides by the wetland with room for improvement with appropriate management.

Organophosphorus Flame Retardants Inhibit Specific Liver Carboxylesterases and Cause Serum Hypertriglyceridemia

PubMed Central

2015-01-01

Humans are prevalently exposed to organophosphorus flame retardants (OPFRs) contained in consumer products and electronics, though their toxicological effects and mechanisms remain poorly understood. We show here that OPFRs inhibit specific liver carboxylesterases (Ces) and cause altered hepatic lipid metabolism. Ablation of the OPFR target Ces1g has been previously linked to dyslipidemia in mice. Consistent with OPFR inhibition of Ces1g, we also observe OPFR-induced serum hypertriglyceridemia in mice. Our findings suggest novel toxicities that may arise from OPFR exposure and highlight the utility of chemoproteomic and metabolomic platforms in the toxicological characterization of environmental chemicals. PMID:24597639

Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

DOEpatents

John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

2016-10-25

A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

Organochlorine and organophosphorus pesticide residues in fodder and milk samples along Musi river belt, India.

PubMed

Kotinagu, Korrapati; Krishnaiah, Nelapati

2015-04-01

The present study was conducted to find the organochlorine pesticide (OCP) and organophosphorus pesticide (OPP) residues in fodder and milk samples along Musi river belt, India. Fodder and milk samples collected from the six zones of Musi river belt, Hyderabad India were analyzed by gas chromatography with electron capture detector for OCP residues and pulsated flame photometric detector for the presence of OPP residues. The gas chromatographic analysis of fodder samples of Zone 5 of Musi river showed the residues of dicofol at concentration of 0.07±0.0007 (0.071-0.077). Among organophosphorus compounds, dimetheoate was present in milk samples collected from Zone 6 at a level of 0.13±0.006 (0.111-0.167). The residues of OCPs, OPPs and cyclodies were below the detection limit in the remaining fodder and milk samples collected from Musi river belt in the present study. The results indicate that the pesticide residues in fodder and milk samples were well below the maximum residue level (MRL) values, whereas dicofol in fodder and dimethoate in milk were slightly above the MRL values specified by EU and CODEX.

[Rapid identification of 22 abused drugs and organophosphorus pesticides in blood by LC-MS/MS].

PubMed

Liu, Hong-tao; Ma, An-de

2009-08-01

To develop a method for rapid identification of 22 abused drugs and organophosphorus pesticides in the blood. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple-reaction monitoring mode (MRM) was employed for detecting the drugs and pesticides in the blood. The MRM database and criteria for identification were established, and ethyl acetate was used for extraction of the drugs. After 3 rounds of extractions of the blood sample (1 mL) using 2 mL ethyl acetate, the extract was vortexed for 3 min and centrifuged at 5000 r/min. Each organic phase was combined and evaporated by gentle N2 gas. The residue was re-dissolved in 100 L mobile phase, from which 5 L was taken for LC-MS/MS detection. The detection of the 22 target compounds could be completed within 10 min. The limit of detection of the target compound ranged from 0.03 to 6.00 ng/ml. Satisfactory results were obtained in proficiency testing program organized by China National Accreditation Service for Conformity Assessment. The method we established is rapid, selective and sensitive for detecting the 22 abused drugs and organophosphorus pesticides.

Organochlorine and organophosphorus pesticide residues in fodder and milk samples along Musi river belt, India

PubMed Central

Kotinagu, Korrapati; Krishnaiah, Nelapati

2015-01-01

Aim: The present study was conducted to find the organochlorine pesticide (OCP) and organophosphorus pesticide (OPP) residues in fodder and milk samples along Musi river belt, India. Materials and Methods: Fodder and milk samples collected from the six zones of Musi river belt, Hyderabad India were analyzed by gas chromatography with electron capture detector for OCP residues and pulsated flame photometric detector for the presence of OPP residues. Results: The gas chromatographic analysis of fodder samples of Zone 5 of Musi river showed the residues of dicofol at concentration of 0.07±0.0007 (0.071-0.077). Among organophosphorus compounds, dimetheoate was present in milk samples collected from Zone 6 at a level of 0.13±0.006 (0.111-0.167). The residues of OCPs, OPPs and cyclodies were below the detection limit in the remaining fodder and milk samples collected from Musi river belt in the present study. Conclusion: The results indicate that the pesticide residues in fodder and milk samples were well below the maximum residue level (MRL) values, whereas dicofol in fodder and dimethoate in milk were slightly above the MRL values specified by EU and CODEX. PMID:27047132

In vitro release of organophosphorus acid anhydrolase from functionalized mesoporous silica against nerve agents.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Baowei; Shah, Saumil S.; Shin, Yongsoon

We report here that under different physiological conditions, biomolecular drugs can be stockpiled in a nanoporous support and afterward can be instantly released when needed for acute responses, and the biomolecular drug molecules can also be gradually released from the nanoporous support over a long time for a complete recovery. Organophosphorus acid anhydrolase (OPAA) was spontaneously and largely entrapped in functionalized mesoporous silica (FMS) due to the dominant electrostatic interaction. The OPAA-FMS composite exhibited a burst release in a pH 9.0 NaHCO(3)-Na(2)CO(3) buffer system and a gradual release in pH 7.4 simulated body fluid. The binding of OPAA to NH(2)-FMSmore » can result in less tyrosinyl and tryptophanyl exposure OPAA molecules to aqueous environment. The bound OPAA in FMS displayed lower activity than the free OPAA in solution prior to the enzyme entrapment. However, the released enzyme maintained the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. The in vitro results in the rabbit serum demonstrate that both OPAA-FMS and the released OPAA may be used as a medical countermeasure against the organophosphorus nerve agents.« less

Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

PubMed Central

Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

2011-01-01

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

PubMed

Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

2011-01-01

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

Organophosphorus insecticide induced decrease in plasma luteinizing hormone concentration in white-footed mice

USGS Publications Warehouse

Rattner, B.A.; Michael, S.D.

1985-01-01

Oral intubation of 50 and 100 mg/kg acephate inhibited brain acetylcholinesterase (AChE) activity by 45% and 56%, and reduced basal luteinizing hormone (LH) concentration by 29% and 25% after 4 h in white-footed mice (Peromyscus leucopus noveboracensis). Dietary exposure to 25, 100, and 400 ppm acephate for 5 days substantially inhibited brain AChE activity, but did not affect plasma LH concentration. These preliminary findings suggest that acute exposure to organophosphorus insecticides may affect LH secretion and possibly reproductive function.

Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights.

PubMed

Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

2016-02-01

The present study investigates the effect of metals on the secretion of enzymes from 12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50% of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.

Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus.

PubMed

Sasaki, Masashi; Takegawa, Kaoru; Kimura, Yoshio

2014-09-17

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn(2+), PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co(2+) PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn(2+) was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co(2+) was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase

Treesearch

Franz St. Johns; Jason C. Hurlbert; John D. Rice; James F. Preston; Edwin Pozharski

2011-01-01

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn by...

The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

PubMed Central

Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

2013-01-01

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design. PMID:23844024

The mode of inhibitor binding to peptidyl-tRNA hydrolase: binding studies and structure determination of unbound and bound peptidyl-tRNA hydrolase from Acinetobacter baumannii.

PubMed

Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2013-01-01

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nomura, Daniel K.; Fujioka, Kazutoshi; Issa, Roger S.

2008-04-01

Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC{sub 50} 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although theremore » was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification.« less

A systematic review on human exposure to organophosphorus pesticides in Iran.

PubMed

Shadboorestan, Amir; Vardanjani, Hossein Molavi; Abdollahi, Mohammad; Goharbari, Mohammad Hadi; Khanjani, Narges

2016-07-02

Human exposure to organophosphorus (OP) pesticides is a serious health challenge. We conducted a systematic review by searching international and national databases for published literature on any human exposure to OPs in Iran from 1990 to March 2015. Qualified papers were in two categories including studies in which biomarkers of exposure were assessed (n = 13; total no. of subjects = 759) and studies that had reported prevalence of OPs-induced poisoning (OPP) and mortality (n = 26; total no. of subjects = 5428). The mean level of activity of acetyl-cholinesterase and butyryl-cholinesterase were 68.65% and 74.2%, respectively. Overall proportion (%) of OPP was estimated (16; 95% CI, 14 to 19).

Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

PubMed

Placido, Antonio; Hai, Tran; Ferrer, Manuel; Chernikova, Tatyana N; Distaso, Marco; Armstrong, Dale; Yakunin, Alexander F; Toshchakov, Stepan V; Yakimov, Michail M; Kublanov, Ilya V; Golyshina, Olga V; Pesole, Graziano; Ceci, Luigi R; Golyshin, Peter N

2015-12-01

A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

Free and ATP-bound structures of Ap4A hydrolase from Aquifex aeolicus V5.

PubMed

Jeyakanthan, Jeyaraman; Kanaujia, Shankar Prasad; Nishida, Yuya; Nakagawa, Noriko; Praveen, Surendran; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

2010-02-01

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 A resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alphabetaalpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

Identification and Characterization of Novel Catalytic Bioscavengers of Organophosphorus Nerve Agents

DTIC Science & Technology

2013-01-01

hydrolase activity . These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had...warfare agents [1–3]. OP nerve agents readily bind covalently to the active site serine in acetylcho- linesterase (AChE), thereby inhibiting the ability...muscarinic receptors, whereas 2-pralidoxime chloride, an oxime nucleophile, reactivates AChE by displacing the phospho- nyl group left on the active site

Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions.

PubMed

Kim, Young-Min; Yamamoto, Eiji; Kang, Min-Sun; Nakai, Hiroyuki; Saburi, Wataru; Okuyama, Masayuki; Mori, Haruhide; Funane, Kazumi; Momma, Mitsuru; Fujimoto, Zui; Kobayashi, Mikihiko; Kim, Doman; Kimura, Atsuo

2012-09-01

Bacteroides thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482) in Escherichia coli and determined that recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization > 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases. © 2012 The Authors Journal compilation © 2012 FEBS.

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

PubMed Central

Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

2008-01-01

Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors. PMID:18097091

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis.

PubMed

Lack, Nathan; Lowe, Edward D; Liu, Jie; Eltis, Lindsay D; Noble, Martin E M; Sim, Edith; Westwood, Isaac M

2008-01-01

Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon-carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 A resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors' knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors.

Structure and substrate-binding mechanism of human Ap4A hydrolase.

PubMed

Swarbrick, James D; Buyya, Smrithi; Gunawardana, Dilantha; Gayler, Kenwyn R; McLennan, Alexander G; Gooley, Paul R

2005-03-04

Asymmetric diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap(4)A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap(4)A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.

Fluorescent Chemosensors for Toxic Organophosphorus Pesticides: A Review

PubMed Central

Obare, Sherine O.; De, Chandrima; Guo, Wen; Haywood, Tajay L.; Samuels, Tova A.; Adams, Clara P.; Masika, Noah O.; Murray, Desmond H.; Anderson, Ginger A.; Campbell, Keith; Fletcher, Kenneth

2010-01-01

Many organophosphorus (OP) based compounds are highly toxic and powerful inhibitors of cholinesterases that generate serious environmental and human health concerns. Organothiophosphates with a thiophosphoryl (P=S) functional group constitute a broad class of these widely used pesticides. They are related to the more reactive phosphoryl (P=O) organophosphates, which include very lethal nerve agents and chemical warfare agents, such as, VX, Soman and Sarin. Unfortunately, widespread and frequent commercial use of OP-based compounds in agricultural lands has resulted in their presence as residues in crops, livestock, and poultry products and also led to their migration into aquifers. Thus, the design of new sensors with improved analyte selectivity and sensitivity is of paramount importance in this area. Herein, we review recent advances in the development of fluorescent chemosensors for toxic OP pesticides and related compounds. We also discuss challenges and progress towards the design of future chemosensors with dual modes for signal transduction. PMID:22163587

Fluorescent chemosensors for toxic organophosphorus pesticides: a review.

PubMed

Obare, Sherine O; De, Chandrima; Guo, Wen; Haywood, Tajay L; Samuels, Tova A; Adams, Clara P; Masika, Noah O; Murray, Desmond H; Anderson, Ginger A; Campbell, Keith; Fletcher, Kenneth

2010-01-01

Many organophosphorus (OP) based compounds are highly toxic and powerful inhibitors of cholinesterases that generate serious environmental and human health concerns. Organothiophosphates with a thiophosphoryl (P=S) functional group constitute a broad class of these widely used pesticides. They are related to the more reactive phosphoryl (P=O) organophosphates, which include very lethal nerve agents and chemical warfare agents, such as, VX, Soman and Sarin. Unfortunately, widespread and frequent commercial use of OP-based compounds in agricultural lands has resulted in their presence as residues in crops, livestock, and poultry products and also led to their migration into aquifers. Thus, the design of new sensors with improved analyte selectivity and sensitivity is of paramount importance in this area. Herein, we review recent advances in the development of fluorescent chemosensors for toxic OP pesticides and related compounds. We also discuss challenges and progress towards the design of future chemosensors with dual modes for signal transduction.

31P-edited diffusion-ordered 1H NMR spectroscopy for the spectral isolation and identification of organophosphorus compounds related to chemical weapons agents and their degradation products.

PubMed

Mayer, Brian P; Valdez, Carlos A; Hok, Saphon; Chinn, Sarah C; Hart, Bradley R

2012-12-04

Organophosphorus compounds represent a large class of molecules that include pesticides, flame-retardants, biologically relevant molecules, and chemical weapons agents (CWAs). The detection and identification of organophosphorus molecules, particularly in the cases of pesticides and CWAs, are paramount to the verification of international treaties by various organizations. To that end, novel analytical methodologies that can provide additional support to traditional analyses are important for unambiguous identification of these compounds. We have developed an NMR method that selectively edits for organophosphorus compounds via (31)P-(1)H heteronuclear single quantum correlation (HSQC) and provides an additional chromatographic-like separation based on self-diffusivities of the individual species via (1)H diffusion-ordered spectroscopy (DOSY): (1)H-(31)P HSQC-DOSY. The technique is first validated using the CWA VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) by traditional two-dimensional DOSY spectra. We then extend this technique to a complex mixture of VX degradation products and identify all the main phosphorus-containing byproducts generated after exposure to a zinc-cyclen organometallic homogeneous catalyst.

Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift in Escherichia coli.

PubMed

Lee, Jae-Woo; Park, Young-Ha; Seok, Yeong-Jae

2018-06-18

Bacteria respond to nutritional stresses by changing the cellular concentration of the alarmone (p)ppGpp. This control mechanism, called the stringent response, depends on two enzymes, the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT in Escherichia coli and related bacteria. Because SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in these bacteria, SpoT activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which is lethal. To date, however, no such regulation of SpoT (p)ppGpp hydrolase activity has been documented in E. coli In this study, we show that Rsd directly interacts with SpoT and stimulates its (p)ppGpp hydrolase activity. Dephosphorylated HPr, but not phosphorylated HPr, of the phosphoenolpyruvate-dependent sugar phosphotransferase system could antagonize the stimulatory effect of Rsd on SpoT (p)ppGpp hydrolase activity. Thus, we suggest that Rsd is a carbon source-dependent regulator of the stringent response in E. coli . Copyright © 2018 the Author(s). Published by PNAS.

USE OF PBPK MODELS FOR ASSESSING ABSORBED DOSE AND CHE INHIBITION FROM AGGREGATE EXPOSURE OF INFANTS AND CHILDREN TO ORGANOPHOSPHORUS INSECTICIDES

EPA Science Inventory

A physiological pharmacokinetic (PBPK) modeling framework has been established to assess cumulative risk of dose and injury of infants and children to organophosphorus (OP) insecticides from aggregate sources and routes. Exposure inputs were drawn from all reasonable sources, pr...

LytN, a Murein Hydrolase in the Cross-wall Compartment of Staphylococcus aureus, Is Involved in Proper Bacterial Growth and Envelope Assembly*

PubMed Central

Frankel, Matthew B.; Hendrickx, Antoni P. A.; Missiakas, Dominique M.; Schneewind, Olaf

2011-01-01

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle. PMID:21784864

DEVELOPMENT OF A RISK ASSESSMENT MODEL FOR THE EFFECTS OF ORGANOPHOSPHORUS PESTICIDES ON INFECTIOUS DISEASE SUSCEPTIBILITY AND THE IMMUNE SYSTEM

EPA Science Inventory

There is increased concern about the sublethal effects of organophosphorus (OP) pesticides on human and animal health. This class of chemicals has been shown to affect the immune function of macrophages and lymphocytes. Malathion, an OP compound, is one of the most widely used ...

Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

USDA-ARS?s Scientific Manuscript database

A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

Folding anomalies of neuroligin3 caused by a mutation in the alpha/beta-hydrolase fold domain.

PubMed

De Jaco, Antonella; Dubi, Noga; Comoletti, Davide; Taylor, Palmer

2010-09-06

Proteins of the alpha/beta-hydrolase fold family share a common structural fold, but perform a diverse set of functions. We have been studying natural mutations occurring in association with congenital disorders in the alpha/beta-hydrolase fold domain of neuroligin (NLGN), butyrylcholinesterase (BChE), acetylcholinesterase (AChE). Starting from the autism-related R451C mutation in the alpha/beta-hydrolase fold domain of NLGN3, we had previously shown that the Arg to Cys substitution is responsible for endoplasmic reticulum (ER) retention of the mutant protein and that a similar trafficking defect is observed when the mutation is inserted at the homologous positions in AChE and BChE. Herein we show further characterization of the R451C mutation in NLGN3 when expressed in HEK-293, and by protease digestion sensitivity, we reveal that the phenotype results from protein misfolding. However, the presence of an extra Cys does not interfere with the formation of disulfide bonds as shown by reaction with PEG-maleimide and estimation of the molecular mass changes. These findings highlight the role of proper protein folding in protein processing and localization. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

FOLDING ANOMALIES OF NEUROLIGIN3 CAUSED BY A MUTATION IN THE α/β-HYDROLASE FOLD DOMAIN

PubMed Central

De Jaco, Antonella; Dubi, Noga; Comoletti, Davide; Taylor, Palmer

2017-01-01

Proteins of the α/β-hydrolase fold family share a common structural fold, but perform a diverse set of functions. We have been studying natural mutations occurring in association with congenital disorders in the α/β-hydrolase fold domain of neuroligin (NLGN), butyrylcholinesterase (BChE), acetylcholinesterase (AChE). Starting from the autism-related R451C mutation in the α/β-hydrolase fold domain of NLGN3, we had previously shown that the Arg to Cys substitution is responsible for endoplasmic reticulum (ER) retention of the mutant protein and that a similar trafficking defect is observed when the mutation is inserted at the homologous positions in AChE and BChE. Herein we show further characterization of the R451C mutation in NLGN3 when expressed in HEK-293, and by protease digestion sensitivity, we reveal that the phenotype results from protein misfolding. However, the presence of an extra Cys doesn’t interfere with the formation of disulfide bonds as shown by reaction with PEG-maleimide and estimation of the molecular mass changes. These findings highlight the role of proper protein folding in protein processing and localization. PMID:20227402

Small-scale purification of butyrylcholinesterase from human plasma and implementation of a μLC-UV/ESI MS/MS method to detect its organophosphorus adducts.

PubMed

John, Harald; Breyer, Felicitas; Schmidt, Christian; Mizaikoff, Boris; Worek, Franz; Thiermann, Horst

2015-10-01

Human butyrylcholinesterase (hBChE) is a serine hydrolase (EC 3.1.1.8) present in all mammalian tissues and the bloodstream. Similar to acetylcholinesterase, the enzyme reacts with organophosphorus compounds (OP) like nerve agents or pesticides that cause enzyme inhibition (BChE adducts). These adducts represent valuable biomarkers for analytical verification of OP exposure. For establishment of these mass spectrometry based methods sufficient amounts of hBChE in high purity are required. Unfortunately, commercial lots are of inappropriate purity thus favouring in-house isolation. Therefore, we developed a small scale procedure to isolate hBChE from citrate plasma. After precipitation by polyethylene glycol (8% w/v and 20% w/v PEG 6000) hBChE was purified from plasma by four consecutive chromatographic steps including anion exchange, affinity extraction and size exclusion. Protein elution was monitored on-line by UV-absorbance (280 nm) followed by continuous fractionation for off-line analysis of (1) hBChE enzyme activity by Ellman assay, (2) protein purity by gel electrophoresis, and (3) protein identity by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Numerous major impurities separated from hBChE were identified. The purified material was used for in vitro incubation with diverse OP to establish a μ-liquid chromatography-ultra violet detection/electrospray ionization tandem-mass spectrometric method (μLC-UV/ESI MS/MS) for detection of hBChE adducts suitable for verification analysis. Analytical data for diverse OP pesticides including deuterated analogues as well as G- and V-type nerve agents and their precursor are summarized. This method was successfully applied to plasma samples provided by the Organisation for the Prohibition of Chemical Weapons (OPCW) for the 4th Biomedical Exercise. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of Bacterial Microflora of the Lower Digestive Tract of Free-Range Waterfowl on Influenza Virus Activation ▿

PubMed Central

King, Marcus D.; Guentzel, M. Neal; Arulanandam, Bernard P.; Bodour, Adria A.; Brahmakshatriya, Vinayak; Lupiani, Blanca; Chambers, James P.

2011-01-01

Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a “trypsin-like” manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established. PMID:21531837

Sol-gel niobia sorbent with a positively charged octadecyl ligand providing enhanced enrichment of nucleotides and organophosphorus pesticides in capillary microextraction for online HPLC analysis.

PubMed

Kesani, Sheshanka; Malik, Abdul

2018-04-01

A niobia-based sol-gel organic-inorganic hybrid sorbent carrying a positively charged C 18 ligand (Nb 2 O 5 -C 18 (+ve)) was synthesized to achieve enhanced enrichment capability in capillary microextraction of organophosphorus compounds (which include organophosphorus pesticides and nucleotides) before their online analysis by high-performance liquid chromatography. The sorbent was designed to simultaneously provide three different types of molecular level interactions: electrostatic, Lewis acid-base, and van der Waals interactions. To understand relative contributions of various molecular level analyte-sorbent interactions in the extraction process, two other sol-gel niobia sorbents were also created: (a) a purely inorganic sol-gel niobia sorbent (Nb 2 O 5 ) and (b) an organic-inorganic hybrid sol-gel niobia sorbent carrying an electrically neutral-bonded octadecyl ligand (Nb 2 O 5 -C 18 ). The extraction efficiency of the created sol-gel niobia sorbent (Nb 2 O 5 -C 18 (+ve)) was compared with that of analogously designed and synthesized titania-based sol-gel sorbent (TiO 2 -C 18 (+ve)), taking into consideration that titania-based sorbents present state-of-the-art extraction media for organophosphorus compounds. In capillary microextraction with high-performance liquid chromatography analysis, Nb 2 O 5 -C 18 (+ve) had shown 40-50% higher specific extraction values (a measure of extraction efficiency) over that of TiO 2 -C 18 (+ve). Compared to TiO 2 -C 18 (+ve), Nb 2 O 5 -C 18 (+ve) also provided superior analyte desorption efficiency (96 vs. 90%) during the online release of the extracted organophosphorus pesticides from the sorbent coating in the capillary microextraction capillary to the chromatographic column using reversed-phase high-performance liquid chromatography mobile phase. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of Organophosphorus Pesticides in Wheat by Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction Combined with HPLC

PubMed Central

Quan, Ji; Hu, Zeshu

2018-01-01

Food safety issues closely related to human health have always received widespread attention from the world society. As a basic food source, wheat is the fundamental support of human survival; therefore, the detection of pesticide residues in wheat is very necessary. In this work, the ultrasonic-assisted ionic liquid-dispersive liquid-liquid microextraction (DLLME) method was firstly proposed, and the extraction and analysis of three organophosphorus pesticides were carried out by combining high-performance liquid chromatography (HPLC). The extraction efficiencies of three ionic liquids with bis(trifluoromethylsulfonyl)imide (Tf2N) anion were compared by extracting organophosphorus in wheat samples. It was found that the use of 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([OMIM][Tf2N]) had both high enrichment efficiency and appropriate extraction recovery. Finally, the method was used for the determination of three wheat samples, and the recoveries of them were 74.8–112.5%, 71.8–104.5%, and 83.8–115.5%, respectively. The results show that the method proposed is simple, fast, and efficient, which can be applied to the extraction of organic matters in wheat samples. PMID:29854562

A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruzzini, Antonio C.; Bhowmik, Shiva; Ghosh, Subhangi

The meta-cleavage product (MCP) hydrolases utilize a Ser–His–Asp triad to hydrolyze a carbon–carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES red, which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES red decay and product formation showed amore » solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pK a2 (β nuc ~ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His–Asp pair does not play an essential role. The data further suggest that ES red represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.« less

Arabidopsis thaliana EPOXIDE HYDROLASE1 (AtEH1) is a cytosolic epoxide hydrolase involved in the synthesis of poly-hydroxylated cutin monomers.

PubMed

Pineau, Emmanuelle; Xu, Lin; Renault, Hugues; Trolet, Adrien; Navrot, Nicolas; Ullmann, Pascaline; Légeret, Bertrand; Verdier, Gaëtan; Beisson, Fred; Pinot, Franck

2017-07-01

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization in vivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaves with constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis. © 2017 CNRS New Phytologist © 2017 New Phytologist Trust.

Inhibition of Phenylamide Hydrolysis by Bacillus sphaericus with Methylcarbamate and Organophosphorus Insecticides

PubMed Central

Engelhardt, G.; Wallnöfer, P. R.

1975-01-01

The degradation of the phenylamide herbicides monolinuron, linuron, and solan by cultures of Bacillus sphaericus ATCC 12123 was inhibited by the methylcarbamate insecticides metmercapturon, aldicarb, propoxur, and carbaryl and by the organophosphorus insecticides fenthion and parathion. The extent of inhibition was largest with metmercapturon and smallest with parathion. Inhibition of hydrolysis of the two phenylurea herbicides was greater than of the acylanilide compound. Tests with crude enzyme preparations of aryl acylamidase derived from B. sphaericus showed that the inhibition of the hydrolysis of linuron with methylcarbamates is a competitive one. The insecticides tested did not induce the enzyme, nor could they serve as its substrate. PMID:1155931

Production of epoxide hydrolases in batch fermentations of Botryosphaeria rhodina.

PubMed

Melzer, Guido; Junne, Stefan; Wohlgemuth, Roland; Hempel, Dietmar C; Götz, Peter

2008-06-01

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

Risk assessment of the cumulative acute exposure of Hungarian population to organophosphorus pesticide residues with regard to consumers of plant based foods.

PubMed

Zentai, Andrea; Szabó, István J; Kerekes, Kata; Ambrus, Árpád

2016-03-01

Based on the Hungarian pesticide residues monitoring data of the last five years and the consumption data collected within a 3-day dietary record survey in 2009 (more than 2 million pesticide residue results and almost 5000, 0-101-year-old consumers 3 non-consecutive-day personal fruit and vegetable consumption data), the cumulative acute exposure of organophosphorus pesticide residues was evaluated. The relative potency factor approach was applied, with acephate chosen as index compound. According to our conservative calculation method, applying the measured residues only, the 99.95% of the 99th percentiles of calculated daily intakes was at or below 87 μg/kgbwday, indicating that the cumulative acute exposure of the whole Hungarian population (including all age classes) to organophosphorus compounds was not a health concern. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro study of the neuropathic potential of the organophosphorus compounds trichlorfon and acephate.

PubMed

Fernandes, Laís S; Emerick, Guilherme L; dos Santos, Neife Aparecida G; de Paula, Eloísa Silva; Barbosa, Fernando; dos Santos, Antonio Cardozo

2015-04-01

Organophosphorus-induced delayed neuropathy (OPIDN) is a central and peripheral distal axonopathy characterized by ataxia and paralysis. Trichlorfon and acephate are two organophosphorus compounds (OPs) used worldwide as insecticide and which cause serious effects to non-target species. Despite that, the neuropathic potential of these OPs remains unclear. The present study addressed the neurotoxic effects and the neuropathic potential of trichlorfon and acephate in SH-SY5Y human neuroblastoma cells, by evaluating inhibition and aging of neuropathy target esterase (NTE), inhibition of acetylcholinesterase (AChE), neurite outgrowth, cytotoxicity and intracellular calcium. Additionally, the effects observed were compared to those of two well-studied OPs: mipafox (known as neuropathic) and paraoxon (known as non-neuropathic). Trichlorfon and mipafox presented the lowest percentage of reactivation of inhibited NTE and the lowest ratio IC50 NTE/IC50 AChE. Moreover, they caused inhibition and aging of at least 70% of the activity of NTE at sub-lethal concentrations. All these effects have been associated with induction of OPIDN. When assayed at these concentrations, trichlorfon and mipafox reduced neurite outgrowth and increased intracellular calcium, events implicated in the development of OPIDN. Acephate caused effects similar to those caused by paraoxon (non-neuropathic OP) and was only able to inhibit 70% of NTE activity at lethal concentrations. These findings suggest that trichlorfon is potentially neuropathic, whereas acephate is not. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metagenomic Functional Potential Predicts Degradation Rates of a Model Organophosphorus Xenobiotic in Pesticide Contaminated Soils

PubMed Central

Jeffries, Thomas C.; Rayu, Smriti; Nielsen, Uffe N.; Lai, Kaitao; Ijaz, Ali; Nazaries, Loic; Singh, Brajesh K.

2018-01-01

Chemical contamination of natural and agricultural habitats is an increasing global problem and a major threat to sustainability and human health. Organophosphorus (OP) compounds are one major class of contaminant and can undergo microbial degradation, however, no studies have applied system-wide ecogenomic tools to investigate OP degradation or use metagenomics to understand the underlying mechanisms of biodegradation in situ and predict degradation potential. Thus, there is a lack of knowledge regarding the functional genes and genomic potential underpinning degradation and community responses to contamination. Here we address this knowledge gap by performing shotgun sequencing of community DNA from agricultural soils with a history of pesticide usage and profiling shifts in functional genes and microbial taxa abundance. Our results showed two distinct groups of soils defined by differing functional and taxonomic profiles. Degradation assays suggested that these groups corresponded to the organophosphorus degradation potential of soils, with the fastest degrading community being defined by increases in transport and nutrient cycling pathways and enzymes potentially involved in phosphorus metabolism. This was against a backdrop of taxonomic community shifts potentially related to contamination adaptation and reflecting the legacy of exposure. Overall our results highlight the value of using holistic system-wide metagenomic approaches as a tool to predict microbial degradation in the context of the ecology of contaminated habitats. PMID:29515526

[The mechanism of the transport of organophosphorus compounds across the histo-hematic barriers].

PubMed

Miroshkina, V N; Kosmachev, A B; Salova, L S

1999-01-01

It was demonstrated in experiments on mice [correction of rats] that the transport of organophosphorus compounds (OPC) through membranes of the histohematic barriers (HHB) of the organism occurs by means of diffusion. The rate of this process depends on the interaction of OPC with the specific sites of binding with the tissues, among which the enzyme carboxylesterase plays an important part. It is suggested that both the rate and direction of OPC diffusion are determined by the relationship between the values of affinity of the ligands for the sites of their specific binding found on both sides of the HHB.

Sorption of Organophosphorus Flame Retardants (OPFRs) on ...

EPA Pesticide Factsheets

Organophosphorus flame retardants (OPFRs) are widely used as additives in industrial and consumer products such as electrical and electronic products, furniture, plastics, textiles, and building/construction materials. Due to human exposure and potential health effects, OPFRs including tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are EPA Action Plan chemicals for chemical assessments under the Toxic Substances Control Act (TSCA). This work investigated the sorption of these three compounds from the air to settled Arizona Test Dust (ATD) and house dust (HD) in a dual small chamber system. The OPFR exposed dust was analyzed to determine the sorption concentration and sorption rate of OPFRs on the dust. The effect of the composition of the dust on OPFR sorption was evaluated. The results showed that ATD and HD have varied sorption capacity for OPFRs from air. This work explores the relationship between OPFR concentrations in settled dust and air. The data can be used to determine partitioning of OPFRs between the gas phase and settled dust indoors and to inform strategies to reduce exposure and risk.

Microchip-Based Organophosphorus Detection Using Bienzyme Bioelectrocatalysis

NASA Astrophysics Data System (ADS)

Han, Yong Duk; Jeong, Chi Yong; Lee, Jun Hee; Lee, Dae-Sik; Yoon, Hyun C.

2012-06-01

We have developed a microsystem for the detection of organophosphorus (OP) compounds using acetylcholine esterase (AchE) and choline oxidase (ChOx) bienzyme bioelectrocatalysis. Because AchE is irreversibly inhibited by OP pesticides, the change in AchE activity with OP treatment can be traced to determine OP concentration. Polymer-associated ChOx immobilization on the working electrode surface and magnetic microparticle (MP)-assisted AchE deposition methods were employed to create an AchE-ChOx bienzyme-modified biosensing system. ChOx was immobilized on the micropatterned electrodes using poly(L-lysine), glutaraldehyde, and amine-rich interfacial surface. AchE was immobilized on the MP surface via Schiff's base formation, and the enzyme-modified MPs were deposited on the working electrode using a magnet under the microfluidic channel. The bioelectrocatalytic reaction between AchE-ChOx bienzyme cascade and the ferrocenyl electron shuttle was successfully used to detect OP with the developed microchip. This provides a self-contained and relatively easy method for OP detection. It requires minimal time and a small sample size, and has potential analytic applications in pesticides and chemical warfare agents.

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun†,‡

PubMed Central

Fleming, Christopher D.; Edwards, Carol C.; Kirby, Stephen D.; Maxwell, Donald M.; Potter, Philip M.; Cerasoli, Douglas M.; Redinbo, Matthew R.

2008-01-01

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 104-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure. PMID:17407327

Down-regulation of anandamide hydrolase in mouse uterus by sex hormones.

PubMed

MacCarrone, M; De Felici, M; Bari, M; Klinger, F; Siracusa, G; Finazzi-Agrò, A

2000-05-01

Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.

Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

PubMed Central

Rodríguez-Durán, Luis V.; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C.; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N.

2011-01-01

Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

DOE PAGES

Lee, Brady D.; Apel, William A.; Sheridan, Peter P.; ...

2018-04-16

Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Here, experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX.

Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Brady D.; Apel, William A.; Sheridan, Peter P.

Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Here, experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX.

Screening of Organophosphorus Acid Anhydrases from different Sources by Western Blot Analysis

DTIC Science & Technology

1991-01-01

agents) have been found3 in both procaryotic and eukaryotic organisms. These enzymes, referred to as organophosphorous acid anhydrases (OPA anhydrases...of organisms, both procaryotic and eukaryotic. Previously, these enzymes were known as DFPases, somanases, paraoxonases or parathion hydrolases, based...cell (SLM Aminco) at 16,000 psi. Cellular debris was removed by centrifugation (46,000xg) for 30 minutes at 40C. The crude cell supernatant which

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

Treesearch

Franz J. St John; Diane Dietrich; Casey Crooks; Edwin Pozharski; Javier M. González; Elizabeth Bales; Kennon Smith; Jason C. Hurlbert

2014-01-01

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the

Recovery of cholinesterase activity in five avian species exposed to dicrotophos, an organophosphorus pesticide

USGS Publications Warehouse

Fleming, W.J.; Grue, C.E.

1981-01-01

The responses of brain and plasma cholinesterase (ChE) activities were examined in mallard ducks, bobwhite quail, barn owls, starlings, and common grackles given oral doses of dicrotophos, an organophosphorus insecticide. Up to an eightfold difference in response of brain ChE activity to dicrotophos was found among these species. Brain ChE activity recovered to within 2 SD of normal within 26 days after being depressed 55 to 64%. Recovery of brain ChE activity was similar among species and followed the model Y = a + b (log10X).

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40

PubMed Central

Lee, Saeyoung; Lee, Jonas Yun; Ha, Sung Chul; Jung, Jina; Shin, Dong Hae; Kim, Kyoung Heon; Choi, In-Geol

2009-01-01

Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl-(1→3)-d-galactose] using various β-agarases. Neoagarobiose hydrolase is an enzyme that acts on the α-­1,3 linkage in neoagarobiose to yield d-galactose and 3,6-anhydro-l-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 Å, β = 101.86°. The crystals diffracted to 1.98 Å resolution and possibly contains two molecules in the asymmetric unit. PMID:20054134

Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

PubMed Central

Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

2016-01-01

Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity*

PubMed Central

Bahl, Christopher D.; Hvorecny, Kelli L.; Bridges, Andrew A.; Ballok, Alicia E.; Bomberger, Jennifer M.; Cady, Kyle C.; O'Toole, George A.; Madden, Dean R.

2014-01-01

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii (“aCif”). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog (“aCifR”) and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections. PMID:24474692

Quantum chemical study of leaving group activation in T. vivax nucleoside hydrolase

NASA Astrophysics Data System (ADS)

Loverix, Stefan; Versees, Wim; Steyaert, Jan; Geerlings, Paul

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. The only groups in the vicinity of the leaving group that contribute to catalysis are (i) the indole side chain of Trp260, and (ii) the 5'-group of the substrate's ribose moiety. The x-ray structure of the slow Asp10Ala mutant of nucleoside hydrolase with the substrate inosine bound in the active site displays a face-to-face aromatic stacking interaction between Trp260 and the purine base of the substrate, as well as a peculiar C4'-endo ribose pucker that allows the 5'-OH group to accept an intramolecular hydrogen bond from the C8 of the purine. The first interaction (aromatic stacking) has been shown to raise the pKa of the leaving purine. Here, we present a DFT study showing that the 5'-OH group of ribose fulfills a similar role, rather than stabilizing the oxocarbenium-like transition state.

Structure of a Trypanosoma Brucei Alpha/Beta--Hydrolase Fold Protein With Unknown Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merritt, E.A.; Holmes, M.; Buckner, F.S.

2009-05-26

The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 {angstrom} using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the {alpha}/{beta}-hydrolase fold family. Structural superposition onto representative {alpha}/{beta}-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similaritymore » at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands {beta}6 and {beta}7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family.« less

Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brzezinski, Krzysztof; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan; Bujacz, Grzegorz

2008-07-01

Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified.more » Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.« less

Emergence of multi drug resistance among soil bacteria exposing to insecticides.

PubMed

Rangasamy, Kirubakaran; Athiappan, Murugan; Devarajan, Natarajan; Parray, Javid A

2017-04-01

Impacts of pesticide exposure on the soil microbial flora and cross resistance to antibiotics have not been well documented. Development of antibiotic resistance is a common issue among soil bacteria which are exposing to pesticides continuously at sub-lethal concentration. The present study was focused to evaluate the correlation between pesticide exposures and evolution of multi drug resistance among isolates collected from soil applied with insecticides. Twenty five insecticide (Monochrotophos) degrading bacteria were isolated from contaminated agricultural soil. The bacterial isolates Bacillus Sps, Bacillus cereus, Bacillus firmus and Bacillus thuringiensis were found to be resistant against chloramphenical, monochrotophos, ampicillin, cefotaxime, streptomycin and tetracycline antibiotics used. Involvement of plasmid in drug as well as insecticide resistant was confirmed through plasmid curing among selected bacterial strains. Bacillus Sps (MK-07), Bacillus cereus (MK-11), Bacillus firmus (MK-13) and Bacillus thuringiensis (MK-24) lost their resistant against insecticides and antibiotics once after removal of plasmid by exposing to 2% sodium dodecyl sulphate. The plasmid was transformed back to bacteria which produced similar derivatives when cultured in Minimal Salt medium (pH 7.0) supplemented with 0.4% of insecticide. Homology modeling was used to prove that organophosphorus hydrolase and able to metabolize all the antibiotics showed positive interaction with high docking score. The present study revealed that persistent of insecticides in the agricultural soil may lead to increasing development of multidrug resistance among soil bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biomarkers of organophosphorus (OP) exposures in humans

PubMed Central

Marsillach, Judit; Richter, Rebecca J.; Kim, Jerry H.; Stevens, Richard C.; MacCoss, Michael J.; Tomazela, Daniela; Suzuki, Stephanie M.; Schopfer, Lawrence M; Lockridge, Oksana; Furlong, Clement E.

2011-01-01

There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of “biomarker proteins” that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS. PMID:21767566

Biomarkers of organophosphorus (OP) exposures in humans.

PubMed

Marsillach, Judit; Richter, Rebecca J; Kim, Jerry H; Stevens, Richard C; MacCoss, Michael J; Tomazela, Daniela; Suzuki, Stephanie M; Schopfer, Lawrence M; Lockridge, Oksana; Furlong, Clement E

2011-10-01

There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS. Copyright © 2011 Elsevier Inc. All rights reserved.

Micellar catalyzed degradation of fenitrothion, an organophosphorus pesticide, in solution and soils.

PubMed

Balakrishnan, Vimal K; Buncel, Erwin; Vanloon, Gary W

2005-08-01

We report on a study of the decomposition of fenitrothion (an organophosphorus pesticide that is a persistent contaminant in soils and groundwater) as catalyzed by cetyltrimethylammonium (CTA+) micelles. The CTA micelles were associated with two types of counterions: (1) inert counterions (e.g. CTABr) and (2) reactive counterions (e.g. CTAOH). The reactive counterion surfactants used were hydroxide anion (HO-) as a normal nucleophile and hydroperoxide anion (HOO-) and the anion of pyruvaldehyde oxime (MINA-) as two alpha-nucleophiles. The reactivity order followed: CTABr < CTAOH < CTAMINA < CTAOOH. Treatment of the rate data using the Pseudo-Phase Ion Exchange (PPIE) model of micellar catalysis showed the ratio k2M/k2w to be less than unity for all the surfactants employed. Rather than arising from a "true catalysis", we attributed the observed rate enhancements to a "concentration effect", where both pesticide and nucleophile were incorporated into the small micellar phase volume. Furthermore, the CTAOOH/CTAOH pair gave an alpha-effect of 57, showing that the alpha-effect can play an important role in micellar systems. We further investigated the effectiveness of reactive counterion surfactants in decontaminating selected environmental solids that were spiked with 27 ppb fenitrothion. The solids were as follows: the clay mineral montmorillonite and SO-1 and S0-2 soils (obtained from the Canadian Certified Reference Materials Project). The reactive counterion surfactant solutions significantly enhanced the rate of fenitrothion degradation in the spiked solids over that obtained when the spiked solid was placed in contact with either 0.02 M KOH or water. The rate enhancements followed the order CTAOOH > CTAMINA approximately CTAOH > KOH > water. We conclude that reactive counterion surfactants, especially with alpha-nucleophiles, hold great potential in terms of remediating soils contaminated by toxic organophosphorus esters.

Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species▿ †

PubMed Central

Beloqui, Ana; Nechitaylo, Taras Y.; López-Cortés, Nieves; Ghazi, Azam; Guazzaroni, María-Eugenia; Polaina, Julio; Strittmatter, Axel W.; Reva, Oleg; Waliczek, Agnes; Yakimov, Michail M.; Golyshina, Olga V.; Ferrer, Manuel; Golyshin, Peter N.

2010-01-01

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales). PMID:20622123

4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

NASA Astrophysics Data System (ADS)

Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

2017-07-01

γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

PubMed

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

Gas chromatography/mass spectrometry comprehensive analysis of organophosphorus, brominated flame retardants, by-products and formulation intermediates in water.

PubMed

Cristale, Joyce; Quintana, Jordi; Chaler, Roser; Ventura, Francesc; Lacorte, Silvia

2012-06-08

A multiresidue method based on gas chromatography coupled to quadrupole mass spectrometry was developed to determine organophosphorus flame retardants, polybromodiphenyl ethers (BDEs 28, 47, 99, 100, 153, 154, 183 and 209), new brominated flame retardants, bromophenols, bromoanilines, bromotoluenes and bromoanisoles in water. Two ionization techniques (electron ionization--EI, and electron capture negative ionization--ECNI) and two acquisition modes (selected ion monitoring--SIM, and selected reaction monitoring--SRM) were compared as regards to mass spectral characterization, sensitivity and quantification capabilities. The highest sensitivity, at expenses of identification capacity, was obtained by GC-ECNI-MS/SIM for most of the compounds analyzed, mainly for PBDEs and decabromodiphenyl ethane while GC-EI-MS/MS in SRM was the most selective technique and permitted the identification of target compounds at the pg level, and identification capabilities increased when real samples were analyzed. This method was further used to evaluate the presence and behavior of flame retardants within a drinking water treatment facility. Organophosphorus flame retardants were the only compounds detected in influent waters at levels of 0.32-0.03 μg L⁻¹, and their elimination throughout the different treatment stages was evaluated. Copyright © 2012 Elsevier B.V. All rights reserved.

Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hughes, Ronny C.; McFeeters, Hana; Coates, Leighton

The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6122 with unit-cell parameters a = b = 63.62,c =more » 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da-1. The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.« less

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates.

PubMed

Seidegård, J; DePierre, J W; Guenthner, T M; Oesch, F

1986-09-01

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were

Age-related pulmonary emphysema in mice lacking alpha/beta hydrolase domain containing 2 gene.

PubMed

Jin, Shoude; Zhao, Gang; Li, Zhenghua; Nishimoto, Yuki; Isohama, Yoichiro; Shen, Jingling; Ito, Takaaki; Takeya, Motohiro; Araki, Kimi; He, Ping; Yamamura, Ken-ichi

2009-03-06

The alpha/beta hydrolase family genes have been identified as down-regulated genes in human emphysematous lungs. Although proteins in the alpha/beta hydrolase family generally act as enzymes, such as lipases, the specific functions of the Abhd2 protein are unknown. To examine the role of Abhd2 in the lung, we analyzed Abhd2 deficient mice obtained by gene trap mutagenesis. Abhd2 was expressed in the alveolar type II cells. Abhd2 deficiency resulted in a decreased level of phosphatidylcholine in the bronchoalveolar lavage. These mice developed spontaneous gradual progression of emphysema, due to increased macrophage infiltration, increased inflammatory cytokines, a protease/anti-protease imbalance and enhanced apoptosis. This phenotype is more akin to the pace of emphysema that develops in humans. Our findings suggest that derangement in alveolar phospholipid metabolism can induce emphysema, and that Abhd2 plays a critical role in maintaining lung structural integrity.

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balotra, Sahil; Newman, Janet; French, Nigel G.

2014-02-19

The amidase domain of the allophanate hydrolase AtzF from Pseudomonas sp. strain ADP has been crystallized and preliminary X-ray diffraction data have been collected. The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P2{sub 1}, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°.

Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'haeseleer, Patrik; Gladden, John M.; Allgaier, Martin

2013-07-19

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus, and for novel populations that are related to thermophilicmore » Paenibacilli and an uncultivated subdivision of the littlestudied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify .3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.« less

Overexpression of Human Senescence Marker Protein 30 in Mice Fails to Offer Protection Against Challenge with Organophosphorus Compounds

DTIC Science & Technology

2011-09-01

poisoning. Toxicology 233: 145-154. 2. Gray AP (1984) Design and structure- activity relationships of antidotes to organophosphorus anticholinesterase ...and is being actively pursued. One approach under investigation is the development of human proteins as bioscavengers that sequester or hydrolyze...the major roles described for SMP30 is in maintaining Ca2+ homeostasis by activating enzymes involved in the regulation of Ca2+ pumps localized in

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

PubMed

Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

2014-08-25

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Diadenosine Tetraphosphate Hydrolase Is Part of the Transcriptional Regulation Network in Immunologically Activated Mast Cells▿

PubMed Central

Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

2008-01-01

We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap4A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap4A, suggesting that Ap4A is a second messenger in this context. For Ap4A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolize Ap4A, we provided here evidence that the “Nudix” type 2 gene product, Ap4A hydrolase, is responsible for Ap4A degradation following the immunological activation of mast cells. The knockdown of Ap4A hydrolase modulated Ap4A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap4A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the β-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap4A as a second messenger in the regulation of gene expression. PMID:18644867

Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells.

PubMed

Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

2008-09-01

We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap(4)A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap(4)A, suggesting that Ap(4)A is a second messenger in this context. For Ap(4)A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolyze Ap(4)A, we provided here evidence that the "Nudix" type 2 gene product, Ap(4)A hydrolase, is responsible for Ap(4)A degradation following the immunological activation of mast cells. The knockdown of Ap(4)A hydrolase modulated Ap(4)A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap(4)A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the beta-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap(4)A as a second messenger in the regulation of gene expression.

A General One-Pot Synthesis of 2H-Indazoles Using an Organophosphorus-Silane System.

PubMed

Schoene, Jens; Bel Abed, Hassane; Schmieder, Peter; Christmann, Mathias; Nazaré, Marc

2018-04-12

A simple and direct approach for the regioselective construction of the privileged 2H-indazole scaffold is described. The developed one-pot strategy employs a phospholene mediated N-N bond formation to access 2H-indazoles. The amount of organophosphorus reagent was minimized by recycling the phospholene oxide with organosilanes as reductant. Starting from functionalized 2-nitrobenzaldehydes and primary amines a mild reductive cyclisation, using commercially available phospholene oxide and silanes, delivered a wide variety of substituted 2H-indazoles in good to excellent yields. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

PubMed

Klein, Isabella; Korber, Martina; Athenstaedt, Karin; Daum, Günther

2017-12-01

In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle. Copyright © 2017 Elsevier B.V. All rights reserved.

Repeated Exposure to Sublethal Doses of the Organophosphorus Compound VX Activates BDNF Expression in Mouse Brain

DTIC Science & Technology

2012-01-01

NUMBER activates BDNF expression in mouse brain 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Pizarro, JM, Chang, WE, Bah, MJ...of the Organophosphorus Compound VX Activates BDNF Expression in Mouse Brain Jose M. Pizarro,*,† Wenling E. Chang,†,‡ Mariama J. Bah,† Linnzi K. M...triphosphate and UTP, and 2 ll modified cytidine triphosphate solution [2mM]), 33P-UTP (specific activity of 5 3 109 cpm/lg), 2 ll RNA polymerase, 2 ll of

Validation of an SPME method, using PDMS, PA, PDMS-DVB, and CW-DVB SPME fiber coatings, for analysis of organophosphorus insecticides in natural waters.

PubMed

Lambropoulou, D A; Sakkas, V A; Albanis, T A

2002-11-01

Solid-phase microextraction (SPME) has been optimized and applied to the determination of the organophosphorus insecticides diazinon, dichlofenthion, parathion methyl, malathion, fenitrothion, fenthion, parathion ethyl, bromophos methyl, bromophos ethyl, and ethion in natural waters. Four types of SPME fiber coated with different stationary phases (PDMS, PA, PDMS-DVB, and CW-DVB) were used to examine their extraction efficiencies for the compounds tested. Conditions that might affect the SPME procedure, such as extraction time and salt content, were investigated to determine the analytical performance of these fiber coatings for organophosphorus insecticides. The optimized procedure was applied to natural waters - tap, sea, river, and lake water - spiked in the concentration range 0.5 to 50 micro g L(-1) to obtain the analytical characteristics. Recoveries were relatively high - >80% for all types of aqueous sample matrix - and the calibration plots were reproducible and linear (R(2)>0.982) for all analytes with all the fibers tested. The limits of detection ranged from 2 to 90 ng L(-1), depending on the detector and the compound investigated, with relative standard deviations in the range 3-15% at all the concentration levels tested. The SPME partition coefficients (K(f)) of the organophosphorus insecticides were calculated experimentally for all the polymer coatings. The effect of organic matter such as humic acids on extraction efficiency was also studied. The analytical performance of the SPME procedure using all the fibers in the tested natural waters proved effective for the compounds.

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†

PubMed Central

Nakagawa, Tetsuto; Shimada, Yoshimi; Pavlova, Nadejda V; Li, Su-Chen; Li, Yu-Teh

2015-01-01

We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S277PDDGKTW and S328TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo2-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5–6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans. PMID:26362869

A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III

PubMed Central

Karačić, Zrinka; Vukelić, Bojana; Ho, Gabrielle H.; Jozić, Iva; Sučec, Iva; Salopek-Sondi, Branka; Kozlović, Marija; Brenner, Steven E.; Ludwig-Müller, Jutta; Abramić, Marija

2017-01-01

In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyses the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase. PMID:27467751

Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure.

PubMed

Nigg, H N; Knaak, J B

2000-01-01

The organophosphorus pesticides of this review were discovered in 1936 during the search for a replacement for nicotine for cockroach control. The basic biochemical characteristics of RBC AChE and BChE were determined in the 1940s. The mechanism of inhibition of both enzymes and other serine esterases was known in the 1940s and, in general, defined in the 1950s. In 1949, the death of a parathion mixer-loader dictated blood enzyme monitoring to prevent acute illness from organophosphorus pesticide intoxication. However, many of the chemical and biochemical steps for serine enzyme inhibition by OP compounds remain unknown today. The possible mechanisms of this inhibition are presented kinetically beginning with simple (by comparison) Michaelis-Menten substrate enzyme interaction kinetics. As complicated as the inhibition kinetics appear here, PBPK model kinetics will be more complex. The determination of inter- and intraindividual variation in RBC ChE and BChE was recognized early as critical knowledge for a blood esterase monitoring program. Because of the relatively constant production of RBCs, variation in RBC AChE was determined by about 1970. The source of plasma (or serum) BChE was shown to be the liver in the 1960s with the change in BChE phenotype to the donor in liver transplant patients. BChE activity was more variable than RBC AChE, and only in the 1990s have BChE individual variation questions been answered. We have reviewed the chemistry, metabolism, and toxicity of organophosphorus insecticides along with their inhibitory action toward tissue acetyl- and butyrylcholinesterases. On the basis of the review, a monitoring program for individuals mixing-loading and applying OP pesticides for commercial applicators was recommended. Approximately 41 OPs are currently registered for use by USEPA in the United States. Under agricultural working conditions, OPs primarily are absorbed through the skin. Liver P-450 isozymes catalyze the desulfurization of

α/β-Hydrolase Domain 6 in the Ventromedial Hypothalamus Controls Energy Metabolism Flexibility.

PubMed

Fisette, Alexandre; Tobin, Stephanie; Décarie-Spain, Léa; Bouyakdan, Khalil; Peyot, Marie-Line; Madiraju, S R Murthy; Prentki, Marc; Fulton, Stephanie; Alquier, Thierry

2016-10-25

α/β-Hydrolase domain 6 (ABHD6) is a monoacylglycerol hydrolase that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Although complete or peripheral ABHD6 loss of function is protective against diet-induced obesity and insulin resistance, the role of ABHD6 in the central control of energy balance is unknown. Using a viral-mediated knockout approach, targeted endocannabinoid measures, and pharmacology, we discovered that mice lacking ABHD6 from neurons of the ventromedial hypothalamus (VMH KO ) have higher VMH 2-AG levels in conditions of endocannabinoid recruitment and fail to physiologically adapt to key metabolic challenges. VMH KO mice exhibited blunted fasting-induced feeding and reduced food intake, energy expenditure, and adaptive thermogenesis in response to cold exposure, high-fat feeding, and dieting (transition to a low-fat diet). Our findings identify ABHD6 as a regulator of the counter-regulatory responses to major metabolic shifts, including fasting, nutrient excess, cold, and dieting, thereby highlighting the importance of ABHD6 in the VMH in mediating energy metabolism flexibility. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

PubMed Central

Ukachukwu, Chiamaka U.; Freeman, Dana M.; Quirk, Stephen

2017-01-01

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition. PMID:28742822

Ecto-diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A)-hydrolase is expressed as an ectoenzyme in a variety of mammalian and human cells and adds new aspects to the turnover of Ap4A.

PubMed

von Drygalski, A; Ogilvie, A

2000-01-01

Ap4A and other dinucleotides participate in the regulation of hemostasis and blood pressure control. With the exception of two previously reported surface anchored ectoAp4A-hydrolases on bovine aortic endothelial and chromaffine cells, all Ap4A-hydrolases reported are intracellular or freely soluble. We demonstrated that ectoAp4A-hydrolases are present on a broad variety of cell types of different species: rat mesangial, bovine corneal epithelial, human Hep-G2 and peridontal cells. Ectoenzyme properties were evaluated on rat mesangium cells. Chromatography of purified plasma membranes on Sephacel 300 resulted in enrichment of ectoAp4A-hydrolase and in separation from ectoATPase. In contrast to ATPase, Ap4A-hydrolase was stable at room temperature. EctoAp4A-hydrolase also recognized ATP as substrate, and therefore is not highly specific. The molecular weight was 180 kD. Unlike ectoAMPase ectoAp4A-hydrolase was not attached via a glycosyl-phosphatidylinositol (GPI)-moiety. Concentrations of PI-PLC 10-100-fold higher than effective for ectoAMPase cleavage (10-100 mU/ml) plus extensively extended incubation times up to eight hours did not result in cleavage of ectoAp4A-hydrolase. The enzyme ectoAp4A-hydrolase might presage a direction for pharmaceutical manipulation in the control of blood pressure and hemostasis.

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis.

PubMed

Negoro, Seiji; Shibata, Naoki; Tanaka, Yusuke; Yasuhira, Kengo; Shibata, Hiroshi; Hashimoto, Haruka; Lee, Young-Ho; Oshima, Shohei; Santa, Ryuji; Oshima, Shohei; Mochiji, Kozo; Goto, Yuji; Ikegami, Takahisa; Nagai, Keisuke; Kato, Dai-Ichiro; Takeo, Masahiro; Higuchi, Yoshiki

2012-02-10

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens.

PubMed

Aylward, Frank O; Burnum, Kristin E; Scott, Jarrod J; Suen, Garret; Tringe, Susannah G; Adams, Sandra M; Barry, Kerrie W; Nicora, Carrie D; Piehowski, Paul D; Purvine, Samuel O; Starrett, Gabriel J; Goodwin, Lynne A; Smith, Richard D; Lipton, Mary S; Currie, Cameron R

2012-09-01

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus-bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

PubMed Central

Aylward, Frank O; Burnum, Kristin E; Scott, Jarrod J; Suen, Garret; Tringe, Susannah G; Adams, Sandra M; Barry, Kerrie W; Nicora, Carrie D; Piehowski, Paul D; Purvine, Samuel O; Starrett, Gabriel J; Goodwin, Lynne A; Smith, Richard D; Lipton, Mary S; Currie, Cameron R

2012-01-01

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans. PMID:22378535

Defining the Estimated Core Genome of Bacterial Populations Using a Bayesian Decision Model

PubMed Central

van Tonder, Andries J.; Mistry, Shilan; Bray, James E.; Hill, Dorothea M. C.; Cody, Alison J.; Farmer, Chris L.; Klugman, Keith P.; von Gottberg, Anne; Bentley, Stephen D.; Parkhill, Julian; Jolley, Keith A.; Maiden, Martin C. J.; Brueggemann, Angela B.

2014-01-01

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance. PMID:25144616

Structure-Function Analysis of a Broad Specificity Populus trichocarpa Endo-β-glucanase Reveals an Evolutionary Link between Bacterial Licheninases and Plant XTH Gene Products*

PubMed Central

Eklöf, Jens M.; Shojania, Shaheen; Okon, Mark; McIntosh, Lawrence P.; Brumer, Harry

2013-01-01

The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. PMID:23572521

Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reindl, W.; Deng, K.; Gladden, J.M.

2011-05-01

The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation ofmore » the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.« less

A Review of Experimental Evidence Linking Neurotoxic Organophosphorus Compounds and Inflammation

PubMed Central

Banks, Christopher N.; Lein, Pamela J.

2012-01-01

Organophosphorus (OP) nerve agents and pesticides inhibit acetylcholinesterase (AChE), and this is thought to be a primary mechanism mediating the neurotoxicity of these compounds. However, a number of observations suggest that mechanisms other than or in addition to AChE inhibition contribute to OP neurotoxicity. There is significant experimental evidence that acute OP intoxication elicits a robust inflammatory response, and emerging evidence suggests that chronic repeated low-level OP exposure also upregulates inflammatory mediators. A critical question that is just beginning to be addressed experimentally is the pathophysiologic relevance of inflammation in either acute or chronic OP intoxication. The goal of this article is to provide a brief review of the current status of our knowledge linking inflammation to OP intoxication, and to discuss the implications of these findings in the context of therapeutic and diagnostic approaches to OP neurotoxicity. PMID:22342984

How Does (E)-2-(Acetamidomethylene)succinate Bind to Its Hydrolase? From the Binding Process to the Final Result

PubMed Central

Zhang, Ji-Long; Zheng, Qing-Chuan; Li, Zheng-Qiang; Zhang, Hong-Xing

2013-01-01

The binding of (E)-2-(acetamidomethylene)succinate (E-2AMS) to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD), steered MD (SMD), and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMS's pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzyme's hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMS's pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism. PMID:23308285

Application of hemoperfusion in severe acute organophosphorus pesticide poisoning

PubMed

Li, Zhenhe; Wang, Guixia; Zhen, Guodong; Zhang, Yuliang; Liu, Jiaqiang; Liu, Shanmei

2017-08-23

Background/aim: The aim of this research is to investigate the clinical efficacy of hemoperfusion in the treatment of severe acute organophosphorus pesticide poisoning (AOPP). Materials and methods: Patients meeting the inclusion criteria were divided into Groups 1 and 2 according to whether hemoperfusion was applied or not. Group 2 was observed as the control group. Conventional therapy for AOPP was given to Groups 1 and 2. Besides conventional treatment, patients in Group 1 were also treated with hemoperfusion therapy. Cholinesterase activity and blood glucose concentration were tested before hemoperfusion and for the first 3 days afterwards. The recovery time of 50% cholinesterase was recorded. At the same time, the incidence and mortality of intermediate syndrome was observed and compared. Results: The incidence and mortality of intermediate syndrome in Group 1 was obviously decreased, and the recovery time of cholinesterase activity was significantly shortened compared with Group 2. Conclusion: Hemoperfusion, used for treating severe AOPP, contributes to the improvement of cholinesterase activity, low incidence and mortality of intermediate syndrome, and increase in curative rate.

Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases

USDA-ARS?s Scientific Manuscript database

We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein...

Drosophila tan Encodes a Novel Hydrolase Required in Pigmentation and Vision

PubMed Central

True, John R; Yeh, Shu-Dan; Hovemann, Bernhard T; Kemme, Tobias; Meinertzhagen, Ian A; Edwards, Tara N; Liou, Shian-Ren; Han, Qian; Li, Jianyong

2005-01-01

Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5′ untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function. PMID:16299587

Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism.

PubMed

Hreljac, Irena; Filipic, Metka

2009-12-01

Organophosphorus compounds (OPs) are widely used as pesticides. They act primarily as neurotoxins, but there is increasing evidence for secondary mechanisms of their toxicity. We have shown that the model OPs, methyl parathion (PT) and methyl paraoxon (PO), are genotoxic. Benzo(a)pyrene (BaP) is a widespread environmental genotoxin found in cigarette smoke, polluted air and grilled food. As people are constantly exposed to low concentrations of BaP and also to OPs, the aim of this study was to determine possible synergistic effects of PT and PO on BaP-induced genotoxicity. In the bacterial reverse mutation assay, PT and PO increased the number of BaP-induced mutations. The comet assay with human hepatoma HepG2 cells showed that BaP-induced DNA strand breaks were increased by PT but slightly decreased by PO. Using the acellular comet assay with UVC-induced DNA strand breaks, we observed a decrease in DNA migration, indicating that OPs cause cross-linking, thus interfering with comet assay results. In HepG2 cells the two OPs induced micronuclei formation at very low doses (0.01 microg/ml) and together with BaP, a more than additive increase of micronuclei was observed, confirming their co-genotoxic effect. We demonstrated for the first time that PT and PO modulate the metabolic activation of BaP. Addition of PT or PO increased aldo-keto reductase (AKR1C1/2) levels in the presence of BaP, while cytochrome 1A (CYP1A) mRNA expression and activity were decreased. Further, specific inhibition of CYP1A had no effect on BaP or OP+BaP-induced micronuclei, whereas inhibition of AKR1C dramatically decreased the number of micronuclei induced by BaP or OP+BaP. Based on these results we propose that co-genotoxicity results from OPs mediated modulation of BaP metabolism, favouring the induction of AKR1C enzymes known to catalyse the formation of DNA reactive BaP o-quinones and the production of reactive oxygen species.

Simple, specific analysis of organophosphorus and carbamate pesticides in sediments using column extraction and gas chromatography

USGS Publications Warehouse

Belisle, A.A.; Swineford, D.M.

1988-01-01

A simple, specific procedure was developed for the analysis of organophosphorus and carbamate pesticides in sediment. The wet soil was mixed with anhydrous sodium sulfate to bind water and the residues were column extracted in acetone:methylene chloride (1:l,v/v). Coextracted water was removed by additional sodium sulfate packed below the sample mixture. The eluate was concentrated and analyzed directly by capillary gas chromatography using phosphorus and nitrogen specific detectors. Recoveries averaged 93 % for sediments extracted shortly after spiking, but decreased significantly as the samples aged.

Simultaneous determination of organophosphorus pesticides in fruits and vegetables using atmospheric pressure gas chromatography quadrupole-time-of-flight mass spectrometry.

PubMed

Cheng, Zhipeng; Dong, Fengshou; Xu, Jun; Liu, Xingang; Wu, Xiaohu; Chen, Zenglong; Pan, Xinglu; Gan, Jay; Zheng, Yongquan

2017-09-15

This paper describes the application of atmospheric pressure gas chromatography quadrupole-time-of-flight mass spectrometry for the simultaneous determination of organophosphorus pesticides in apple, pear, tomato, cucumber and cabbage. Soft ionization with atmospheric pressure ionization source was compared with traditional electron impact ionization (EI). The sensitivity of GC coupled to atmospheric pressure ionization (APGC) for all the analytes was enhanced by 1.0-8.2 times. The ionization modes with atmospheric pressure ionization source was studied by comparing the charge-transfer and proton-transfer conditions. The optimized QuEChERs method was used to pretreat the samples. The calibration curves were found linear from 10 to 1000μg/L, obtaining correlation coefficients higher than 0.9845. Satisfactory mean recovery values, in the range of 70.0-115.9%, and satisfactory precision, with all RSD r <19.7% and all RSD R values <19.5% at the three fortified concentration levels for all the fifteen OPPs. The results demonstrate the potential of APGC-QTOF-MS for routine quantitative analysis of organophosphorus pesticide in fruits and vegetables. Copyright © 2017 Elsevier Ltd. All rights reserved.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew

The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence ofmore » replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.« less

Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury

PubMed Central

Liu, Hao; Li, Wenjin; Ahmad, Muzamil; Miller, Tricia M.; Rose, Marie E.; Poloyac, Samuel M.; Uechi, Guy; Balasubramani, Manimalha; Hickey, Robert W.; Graham, Steven H.

2010-01-01

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), are active prostaglandin metabolites exerting a variety of biological effects that may be important in the pathogenesis of neurological diseases. Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain specific deubiquitinating enzyme whose aberrant function has been linked to neurodegenerative disorders. We report that [15d-PGJ2] detected by quadrapole mass spectrometry (MS) increases in rat brain after temporary focal ischemia, and that treatment with 15d-PGJ2 induces accumulation of ubiquitinated proteins and exacerbates cell death in normoxic and hypoxic primary neurons. 15d-PGJ2 covalently modifies UCH-L1 and inhibits its hydrolase activity. Pharmacologic inhibition of UCH-L1 exacerbates hypoxic neuronal death while transduction with a TAT-UCH-L1 fusion protein protects neurons from hypoxia. These studies indicate UCH-L1 function is important in hypoxic neuronal death and excessive production of CyPGs after stroke may exacerbate ischemic injury by modification and inhibition of UCH-L1. PMID:20933087

[Research on rapid and quantitative detection method for organophosphorus pesticide residue].

PubMed

Sun, Yuan-Xin; Chen, Bing-Tai; Yi, Sen; Sun, Ming

2014-05-01

The methods of physical-chemical inspection is adopted in the traditional pesticide residue detection, which require a lot of pretreatment processes, are time-consuming and complicated. In the present study, the authors take chlorpyrifos applied widely in the present agricultural field as the research object and propose a rapid and quantitative detection method for organophosphorus pesticide residues. At first, according to the chemical characteristics of chlorpyrifos and comprehensive chromogenic effect of several colorimetric reagents and secondary pollution, the pretreatment of the scheme of chromogenic reaction of chlorpyrifos with resorcin in a weak alkaline environment was determined. Secondly, by analyzing Uv-Vis spectrum data of chlorpyrifos samples whose content were between 0. 5 and 400 mg kg-1, it was confirmed that the characteristic information after the color reaction mainly was concentrated among 360 approximately 400 nm. Thirdly, the full spectrum forecasting model was established based on the partial least squares, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 995 6, standard deviation of calibration (RMSEC) was 2. 814 7 mg kg-1, and standard deviation of verification (RMSEP) was 8. 012 4 mg kg-1. Fourthly, the wavelengths whose center wavelength is 400 nm was extracted as characteristic region to build a forecasting model, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 999 3, standard deviation of calibration (RMSEC) was 2. 566 7 mg kg-1 , standard deviation of verification (RMSEP) was 4. 886 6 mg kg-1, respectively. At last, by analyzing the near infrared spectrum data of chlorpyrifos samples with contents between 0. 5 and 16 mg kg-1, the authors found that although the characteristics of the chromogenic functional group are not obvious, the change of absorption peaks of resorcin itself in the neighborhood of 5 200 cm

CELL SURFACE DISPLAY OF ORGANOPHOSPHORUS HYDROLASE USING ICE NUCLEATION PROTEIN. (R827227)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

PubMed Central

Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

2015-01-01

Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

USDA-ARS?s Scientific Manuscript database

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphoro thioyloxy) benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-l...

Acetylcholinesterase-reduced graphene oxide hybrid films for organophosphorus neurotoxin sensing via quartz crystal microbalance

NASA Astrophysics Data System (ADS)

Tang, Shi; Ma, Wenying; Xie, Guangzhong; Su, Yuanjie; Jiang, Yadong

2016-09-01

An acetylcholinesterase (AChE)-reduced graphene oxide (RGO) hybrid films based biosensor enabled by quartz crystal microbalance (QCM) has been developed for the detection of organophosphorus neurotoxin in gas phase at room temperature. To improve the sensing performance, RGO was used to immobilize large quantities of enzyme and provide a favorable microenvironment to maintain the enzyme activity. The experimental results reveal that the response of AChE-RGO/glutaraldehyde based sensors is about 8 times larger than that of the AChE with the sensitivity of 1.583 Hz/mg/m3. 1.0 mg amount of RGO, 5% concentration of glutaraldehyde and pH 6.8 is the optimal condition of this biosensor.

[Rapid simultaneous determination of organophosphorus pesticides in human serum and urine by liquid chromatography-mass spectrometry].

PubMed

Zlatković, Milica; Jovanović, Miodrag; Djordjević, Dragana; Vucinić, Slavica

2010-09-01

Analysis of organophosphosphorus compounds and their metabolites in a biological material includes the use of numerous methods, covering both preparation of samples for analysis and their identification that is considered to be very complex. Low concentrations monitoring requires implementation of highly sensitive analytical techniques. The aim of this study was to develop and validate an original and sensitive method for the detection and quantitation of organophosphorus pesticides (dimethoate, diazinon, malathion and malaoxon) in human biological matrices (serum, urine). This method was based on a solid-phase extraction procedure, a chromatographic separation using an ACQUITY UPLC HSST3 column and mass spectrometric detection in the positive ion mode. Mobile phase: was consited of Solvent A (5 mM ammonium formate pH 3.0) and Solvent B (0.1% acetic formate in methanol), in a linear gradient (constant flow-rate 0.3 mL/min). The standard curve was linear in the range of 0.05-5.00 mg/L for malathion and malaoxon, 0.10-5.00 mg/L for dimethoate and 0.05-2.50 mg/L for diazinon. The correlation coefficient was r > or = 0.99. Extraction recoveries were satisfactory and ranged between 90-99%. The limits of detection (LOD) was between 0.007-0.07 mg/L and the limits of quantitation (LOQ) ranged between 0.022-0.085 mg/L. Intra- and interassay precision and accuracy were satisfactory for all of the pesticides analyzed. The method of liquid chromatography-mass spectrometry is simple, accurate, and useful for the determination of organophosphorus pesticides in both clinical and forensic toxicology.

Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases.

PubMed

Kim, Ji Young; Jeong, Hyung Jae; Park, Ji-Young; Kim, Young Min; Park, Su-Jin; Cho, Jung Keun; Park, Ki Hun; Ryu, Young Bae; Lee, Woo Song

2012-03-01

Sialidases are enzymes that catalyze the hydrolysis of sialic acid residues from various glycoconjugates, which are widely found in a number of viral and microbial pathogens. In this study, we investigated the biological evaluation of isolated six shikonins (1-6) and three shikonofurans (7-9) from Lithospermum erythrorhizon. The nine isolated compounds 1-9 showed strong and selective inhibition of glycosyl hydrolase (GH) 33 and -34 sialidases activities. In GH33 bacterial-sialidase inhibition assay, the inhibitory activities against GH33 siadliase of all shikonofuran derivatives (7-9) were greater than shikonin derivatives (1-6). Shikonofuran E (8) exhibited the most potent inhibitory activity toward GH33 sialidases (IC(50)=0.24μM). Moreover, our detailed kinetic analysis of these species unveiled that they are all competitive and simple reversible slow-binding inhibitors. Otherwise, they showed different inhibitory capacities and kinetic modes to GH34 viral-sialidase activity. All the naphthoquinone derivatives (1-6) were of almost equal efficiency with IC(50) value of 40μM and shikonofurans (7-9) did not show the significant inhibitory effect to GH34 sialidase. Kinetic analyses indicated that naphthoquinones acted via a noncompetitive mechanism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biosynthesis, structural architecture and biotechnological potential of bacterial tannase: a molecular advancement.

PubMed

Jana, Arijit; Halder, Suman Kumar; Banerjee, Amrita; Paul, Tanmay; Pati, Bikash Ranjan; Mondal, Keshab Chandra; Das Mohapatra, Pradeep Kumar

2014-04-01

Tannin-rich materials are abundantly generated as wastes from several agroindustrial activities. Therefore, tannase is an interesting hydrolase, for bioconversion of tannin-rich materials into value added products by catalyzing the hydrolysis of ester and depside bonds and unlocked a new prospect in different industrial sectors like food, beverages, pharmaceuticals, etc. Microorganisms, particularly bacteria are one of the major sources of tannase. In the last decade, cloning and heterologous expression of novel tannase genes and structural study has gained momentum. In this article, we have emphasized critically on bacterial tannase that have gained worldwide research interest for their diverse properties. The present paper delineate the developments that have taken place in understanding the role of tannase action, microbial sources, various cultivation aspects, downstream processing, salient biochemical properties, structure and active sites, immobilization, efforts in cloning and overexpression and with special emphasis on recent molecular and biotechnological achievements. Copyright © 2014 Elsevier Ltd. All rights reserved.

Supercritical Fluid Extraction of Toxic Heavy Metals and Uranium from Acidic Solutions with Sulfur-Containing Organophosphorus Reagents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yuehe; Liu, Chongxuan; Wu, Hong

2003-03-02

The feasibility of using sulfur-containing organophosphorus reagents for the chelation-supercritical fluid extraction (SFE) of toxic heavy metals and uranium from acidic media was investigated. The SFE experiments were conducted in a specially-designed flow-through liquid extractor. Effective extraction of the metal ions from various acidic media was demonstrated. The effect of ligand concentration in supercritical CO{sub 2} on the kinetics of metal extraction was studied. A simplified model is used to describe the extraction kinetics and the good agreement of experimental data with the equilibrium-based model is achieved.

Application of brain cholinesterase reactivation to differentiate between organophosphorus and carbamate pesticide exposure in wild birds

USGS Publications Warehouse

Smith, M.R.; Thomas, N.J.; Hulse, C.

1995-01-01

Brain cholinesterase activity was measured to evaluate pesticide exposure in wild birds. Thermal reactivation of brain cholinesterase was used to differentiate between carbamate and organophosphorus pesticide exposure. Brain cholinesterase activity was compared with gas chromatography and mass spectrometry of stomach contents. Pesticides were identified and confirmed in 86 of 102 incidents of mortality from 29 states within the USA from 1986 through 1991. Thermal reactivation of cholinesterase activity was used to correctly predict carbamates in 22 incidents and organophosphates in 59 incidents. Agreement (P < 0.001) between predictions based on cholinesterase activities and GC/MS results was significant.

A systems-level approach for investigating organophosphorus pesticide toxicity.

PubMed

Zhu, Jingbo; Wang, Jing; Ding, Yan; Liu, Baoyue; Xiao, Wei

2018-03-01

The full understanding of the single and joint toxicity of a variety of organophosphorus (OP) pesticides is still unavailable, because of the extreme complex mechanism of action. This study established a systems-level approach based on systems toxicology to investigate OP pesticide toxicity by incorporating ADME/T properties, protein prediction, and network and pathway analysis. The results showed that most OP pesticides are highly toxic according to the ADME/T parameters, and can interact with significant receptor proteins to cooperatively lead to various diseases by the established OP pesticide -protein and protein-disease networks. Furthermore, the studies that multiple OP pesticides potentially act on the same receptor proteins and/or the functionally diverse proteins explained that multiple OP pesticides could mutually enhance toxicological synergy or additive on a molecular/systematic level. To the end, the integrated pathways revealed the mechanism of toxicity of the interaction of OP pesticides and elucidated the pathogenesis induced by OP pesticides. This study demonstrates a systems-level approach for investigating OP pesticide toxicity that can be further applied to risk assessments of various toxins, which is of significant interest to food security and environmental protection. Copyright © 2017 Elsevier Inc. All rights reserved.

Zero valent Fe-reduced graphene oxide quantum dots as a novel magnetic dispersive solid phase microextraction sorbent for extraction of organophosphorus pesticides in real water and fruit juice samples prior to analysis by gas chromatography-mass spectrometry.

PubMed

Akbarzade, Samaneh; Chamsaz, Mahmoud; Rounaghi, Gholam Hossein; Ghorbani, Mahdi

2018-01-01

A selective and sensitive magnetic dispersive solid-phase microextraction (MDSPME) coupled with gas chromatography-mass spectrometry was developed for extraction and determination of organophosphorus pesticides (Sevin, Fenitrothion, Malathion, Parathion, and Diazinon) in fruit juice and real water samples. Zero valent Fe-reduced graphene oxide quantum dots (rGOQDs@ Fe) as a new and effective sorbent were prepared and applied for extraction of organophosphorus pesticides using MDSPME method. In order to study the performance of this new sorbent, the ability of rGOQDs@ Fe was compared with graphene oxide and magnetic graphene oxide nanocomposite by recovery experiments of the organophosphorus pesticides. Several affecting parameters in the microextraction procedure, including pH of donor phase, donor phase volume, stirring rate, extraction time, and desorption conditions such as the type and volume of solvents and desorption time were thoroughly investigated and optimized. Under the optimal conditions, the method showed a wide linear dynamic range with R-square between 0.9959 and 0.9991. The limit of detections, the intraday and interday relative standard deviations (n = 5) were less than 0.07 ngmL -1 , 4.7, and 8.6%, respectively. The method was successfully applied for extraction and determination of organophosphorus pesticides in real water samples (well, river and tap water) and fruit juice samples (apple and grape juice). The obtained relative recoveries were in the range of 82.9%-113.2% with RSD percentages of less than 5.8% for all the real samples.

Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

PubMed

Schinke, Claudia; Germani, José C

2012-03-01

Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT JUVENILE HORMONE EPOXIDE HYDROLASE (JHEH) FROM MANDUCA SEXTA. (R825433)

EPA Science Inventory

The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activit...

Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

PubMed

Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

2016-01-01

This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

PubMed

Tsujita, Natsumi; Kuwahara, Hiroyuki; Koyama, Hiroki; Yanaka, Noriyuki; Arakawa, Kenji; Kuniyoshi, Hisato

2017-05-01

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

Sorption of Organophosphorus Flame-Retardants on Settled ...

EPA Pesticide Factsheets

Dust is an important sink for indoor air pollutants, such as organophosphorus flame-retardants (OPFRs) that are used as additives in industrial and consumer products including electrical and electronic products, furniture, plastics, textile, and building/construction materials. This research investigated the sorption of OPFRs, tris(2-chloroethyl) phosphate (TCEP), tris(1-chlor-2-propyl) phosphate (TCPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) on settled Arizona Test Dust (ATD) using a dual small chamber system. During the test, seven free film release paper dust trays covered with ATD were placed in the sink test chamber. Constant gas phase OPFRs from the source chamber were dosed into the test chamber. The dust evenly spread on each dust tray was removed from the test chamber at different exposure times to determine the amount of OPFRs absorbed by the dust. The ATD has been characterized for a nominal particle size and surface area. The mass of dust on each of seven dust trays was weighed before the dust was placed inside the sink chamber. OPFRs concentrations at the inlet and faceplate of the test chamber were monitored by collecting polyurethane foam (PUF) samples. The OPFR exposed dust and PUF samples were extracted by 1:1 ethyl acetate/methylene chloride and analyzed on GC/MS. The data were used to calculate the OPFR sorption concentration on the dust through dust/air partition. Settled dust can adsorb OPFR from air. The sorption concentration wa

Household organophosphorus pesticide use and Parkinson's disease.

PubMed

Narayan, Shilpa; Liew, Zeyan; Paul, Kimberly; Lee, Pei-Chen; Sinsheimer, Janet S; Bronstein, Jeff M; Ritz, Beate

2013-10-01

Household pesticide use is widespread in the USA. Since the 1970s, organophosphorus chemicals (OPs) have been common active ingredients in these products. Parkinson's disease (PD) has been linked to pesticide exposures but little is known about the contributions of chronic exposures to household pesticides. Here we investigate whether long-term use of household pesticides, especially those containing OPs, increases the odds of PD. In a population-based case-control study, we assessed frequency of household pesticide use for 357 cases and 807 controls, relying on the California Department of Pesticide Regulation product label database to identify ingredients in reported household pesticide products and the Pesticide Action Network pesticide database of chemical ingredients. Using logistic regression we estimated the effects of household pesticide use. Frequent use of any household pesticide increased the odds of PD by 47% [odds ratio (OR)=1.47, (95% confidence interval (CI): 1.13, 1.92)]; frequent use of products containing OPs increased the odds of PD more strongly by 71% [OR=1.71, (95% CI: 1.21, 2.41)] and frequent organothiophosphate use almost doubled the odds of PD. Sensitivity analyses showed that estimated effects were independent of other pesticide exposures (ambient and occupational) and the largest odds ratios were estimated for frequent OP users who were carriers of the 192QQ paraoxonase genetic variant related to slower detoxification of OPs. We provide evidence that household use of OP pesticides is associated with an increased risk of developing PD.

Organophosphorus esters in the oceans and possible relation with ocean gyres.

PubMed

Cheng, Wenhan; Xie, Zhouqing; Blais, Jules M; Zhang, Pengfei; Li, Ming; Yang, Chengyun; Huang, Wen; Ding, Rui; Sun, Liguang

2013-09-01

Four organophosphorus esters (OPEs) were detected in aerosol samples collected in the West Pacific, the Indian Ocean and the Southern Ocean from 2009 to 2010, suggesting their circumpolar and global distribution. In general, the highest concentrations were detected near populated regions in China, Australia and New Zealand. OPE concentrations in the Southern Ocean were about two orders of magnitude lower than those near major continents. Additionally, relatively high OPE concentrations were detected at the Antarctic Peninsula, where several scientific survey stations are located. The four OPEs investigated here are significantly correlated with each other, suggesting they may derive from the same source. In the circumpolar transect, OPE concentrations were associated with ocean gyres in the open ocean. Their concentrations were positively related with average vorticity in the sampling area suggesting that a major source of OPEs may be found in ocean gyres where plastic debris is known to accumulate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pharmacotherapy to protect the neuromuscular junction after acute organophosphorus pesticide poisoning.

PubMed

Bird, Steven B; Krajacic, Predrag; Sawamoto, Keigo; Bunya, Naofumi; Loro, Emanuele; Khurana, Tejvir S

2016-06-01

Organophosphorus (OP) pesticide poisoning is a leading cause of morbidity and mortality in the developing world, affecting an estimated three million people annually. Much of the morbidity is directly related to muscle weakness, which develops 1-4 days after poisoning. This muscle weakness, termed the intermediate syndrome (IMS), leads to respiratory, bulbar, and proximal limb weakness and frequently necessitates the use of mechanical ventilation. While not entirely understood, the IMS is most likely due to persistently elevated acetylcholine (ACh), which activates nicotinic ACh receptors at the neuromuscular junction (NMJ). Thus, the NMJ is potentially a target-rich area for the development of new therapies for acute OP poisoning. In this manuscript, we discuss what is known about the IMS and studies investigating the use of nicotinic ACh receptor antagonists to prevent or mitigate NMJ dysfunction after acute OP poisoning. © 2016 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol, Nivalenol, and HT-2 Toxin in Cereal Matrices

PubMed Central

Varga, Elisabeth; Malachova, Alexandra; Nguyen, Nhung Thi; Lorenz, Cindy; Haltrich, Dietmar; Berthiller, Franz; Adam, Gerhard

2015-01-01

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min−1 mg−1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples. PMID:25979885

Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

PubMed

Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

2014-11-01

Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis

PubMed Central

Guo, Yongxia; Zheng, Zuyu; La Clair, James J.; Chory, Joanne; Noel, Joseph P.

2013-01-01

Genetic studies in Arabidopsis implicate an α/β-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-promoting butenolide small molecules found in the smoke of burned plants. However, direct biochemical evidence for the interaction between KAI2 and karrikin and for the mechanism of downstream signaling by a KAI2–karrikin complex remain elusive. We report crystallographic analyses and ligand-binding experiments for KAI2 recognition of karrikins. The karrikin-1 (KAR1) ligand sits in the opening to the active site abutting a helical domain insert but distal from the canonical catalytic triad (Ser95-His246-Asp217) of α/β-hydrolases, consistent with the lack of detectable hydrolytic activity by purified KAI2. The closest approach of KAR1 to Ser95-His246-Asp217 is 3.8 Å from His246. Six aromatic side chains, including His246, encapsulate KAR1 through geometrically defined aromatic–aromatic interactions. KAR1 binding induces a conformational change in KAI2 at the active site entrance. A crevice of hydrophobic residues linking the polar edge of KAR1 and the helical domain insert suggests that KAI2–KAR1 creates a contiguous interface for binding signaling partners in a ligand-dependent manner. PMID:23613584

Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis.

PubMed

Guo, Yongxia; Zheng, Zuyu; La Clair, James J; Chory, Joanne; Noel, Joseph P

2013-05-14

Genetic studies in Arabidopsis implicate an α/β-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-promoting butenolide small molecules found in the smoke of burned plants. However, direct biochemical evidence for the interaction between KAI2 and karrikin and for the mechanism of downstream signaling by a KAI2-karrikin complex remain elusive. We report crystallographic analyses and ligand-binding experiments for KAI2 recognition of karrikins. The karrikin-1 (KAR1) ligand sits in the opening to the active site abutting a helical domain insert but distal from the canonical catalytic triad (Ser95-His246-Asp217) of α/β-hydrolases, consistent with the lack of detectable hydrolytic activity by purified KAI2. The closest approach of KAR1 to Ser95-His246-Asp217 is 3.8 Å from His246. Six aromatic side chains, including His246, encapsulate KAR1 through geometrically defined aromatic-aromatic interactions. KAR1 binding induces a conformational change in KAI2 at the active site entrance. A crevice of hydrophobic residues linking the polar edge of KAR1 and the helical domain insert suggests that KAI2-KAR1 creates a contiguous interface for binding signaling partners in a ligand-dependent manner.

Leaching of the organophosphorus nematicide fosthiazate.

PubMed

Karpouzas, Dimitrios G; Pantelelis, Ioannis; Menkissoglu-Spiroudi, Urania; Golia, Evangelia; Tsiropoulos, Nikolas G

2007-07-01

Fosthiazate is an organophosphorus nematicide which was recently included in Annex I of the Directive 91/414/EEC under the clause that it should be used with special care in soils vulnerable to leaching. Thus, the leaching of fosthiazate was investigated in columns packed with three different soils which represented situations of high (site 2), intermediate (site 1) and low (site 3) leaching potential. The recommended dose of fosthiazate was applied at the surface of the soil columns and fosthiazate fate and transport was investigated for the next two months. Fosthiazate concentrations in the leachate collected from the bottom of the columns packed with soil from site 2 exceeded 0.1 microgl(-1) in most cases. This soil was characterized as acidic, indicating longer fosthiazate persistence, with low organic matter content, indicating weak adsorption, thus representing a situation vulnerable to leaching. In contrast, the lowest concentrations of fosthiazate in the leachate were evident in the columns packed with soil from site 3. This soil was characterized as alkaline, indicating faster degradation, with higher organic matter content, indicating stronger adsorption, thus representing a situation not favoring leaching of fosthiazate. The highest concentration of fosthiazate in the leachate from the columns packed with soil from site 2 was 3.44 microgl(-1) compared to 1.17 and 0.16 microgl(-1), which were the corresponding maximum values measured in columns packed with soil from sites 1 and 3, respectively. The results of the current study further suggest that fosthiazate is mobile in soil and can leach under conducive soil conditions like acidic soils with low organic matter content.

[Soil hydrolase characteristics in late soil-thawing period in subalpine/alpine forests of west Sichuan].

PubMed

Tan, Bo; Wu, Fu-Zhong; Yang, Wan-Qin; Yu, Sheng; Yang, Yu-Lian; Wang, Ao

2011-05-01

Late soil-thawing period is a critical stage connecting winter and growth season. The significant temperature fluctuation at this stage might have strong effects on soil ecological processes. In order to understand the soil biochemical processes at this stage in the subalpine/alpine forests of west Sichuan, soil samples were collected from the representative forests including primary fir forest, fir and birch mixed forest, and secondary fir forest in March 5-April 25, 2009, with the activities of soil invertase, urease, and phosphatase (neutral, acid and alkaline phosphatases) measured. In soil frozen period, the activities of the three enzymes in test forests still kept relatively higher. With the increase of soil temperature, the activities of hydrolases at the early stage of soil-thawing decreased rapidly after a sharp increase, except for neutral phosphatease. Thereafter, there was an increase in the activities of urease and phosphatase. Relative to soil mineral layer, soil organic layer had higher hydrolase activity in late soil-thawing period, and showed more obvious responses to the variation of soil temperature.

A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

PubMed Central

Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

2016-01-01

Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vose, Sarah C.; Center for Children's Environmental Health Research, School of Public Health, University of California, Berkeley, CA 94720; Fujioka, Kazutoshi

2008-11-01

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC ormore » phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.« less

Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing

PubMed Central

Porter, Joanne L.; Carr, Paul D.; Collyer, Charles A.; Ollis, David L.

2014-01-01

Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/β-hydrolase fold structure. It readily crystallizes in space group P212121 from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85 Å resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein–protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices. PMID:25005082

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

PubMed

Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

2008-02-01

The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

ERIC Educational Resources Information Center

Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

2009-01-01

Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

PubMed Central

Gallie, D R; Chang, S C

1997-01-01

Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources. PMID:9414556

Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

PubMed

Gallie, D R; Chang, S C

1997-12-01

Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources.

A SAW-based chemical sensor for detecting sulfur-containing organophosphorus compounds using a two-step self-assembly and molecular imprinting technology.

PubMed

Pan, Yong; Yang, Liu; Mu, Ning; Shao, Shengyu; Wang, Wen; Xie, Xiao; He, Shitang

2014-05-19

This paper presents a new effective approach for the sensitive film deposition of surface acoustic wave (SAW) chemical sensors for detecting organophosphorus compounds such as O-ethyl-S-2-diisopropylaminoethyl methylphosphonothiolate (VX) containing sulfur at extremely low concentrations. To improve the adsorptive efficiency, a two-step technology is proposed for the sensitive film preparation on the SAW delay line utilizing gold electrodes. First, mono[6-deoxy-6-[(mercaptodecamethylene)thio

A SAW-Based Chemical Sensor for Detecting Sulfur-Containing Organophosphorus Compounds Using a Two-Step Self-Assembly and Molecular Imprinting Technology

PubMed Central

Pan, Yong; Yang, Liu; Mu, Ning; Shao, Shengyu; Wang, Wen; Xie, Xiao; He, Shitang

2014-01-01

This paper presents a new effective approach for the sensitive film deposition of surface acoustic wave (SAW) chemical sensors for detecting organophosphorus compounds such as O-ethyl-S-2-diisopropylaminoethyl methylphosphonothiolate (VX) containing sulfur at extremely low concentrations. To improve the adsorptive efficiency, a two-step technology is proposed for the sensitive film preparation on the SAW delay line utilizing gold electrodes. First, mono[6-deoxy-6-[(mercaptodecamethylene)thio

Investigation of the Germination of Barley and Wheat Grains with a Design of Experiments for the Production of Hydrolases

PubMed Central

Kranz, Bertolt; Koch, Milena; Schapfl, Matthias

2015-01-01

Summary The production of hydrolases from cereals has been examined in order to investigate food-derived enzymes as an alternative source to microbial enzymes for the use in food processes. For that, the influence of temperature on the pretreatment, imbibition and germination of barley and wheat grains was determined by measuring the β-glucosidase, β-galactosidase and lipase activities using a design of experiments. The evaluation of the statistical model showed an increase of the β-glucosidase activity with low imbibition and low germination temperature for barley grains and low imbibition and high germination temperature for wheat grains. The maximum β-glucosidase activity in wheat extracts was (585±151) nkat per g of dry mass (dm), while in barley extracts it was (109±15) nkat per g of dm. The maximum β-galactosidase activities in barley and wheat extracts were (34±12) and (63±23) nkat per g of dm, respectively. The maximum lipase activities of (6.7±0.1) and (4.6±4.4) nkat per g of dm in barley and wheat extracts, respectively, were rather low compared to the glycosidase activities. The extracts were also tested for other hydrolase activities (e.g. peptidase and α-amylase activities). The insights obtained enable the basis for the potential use of cereal hydrolases in food processing, which might be attractive to consumers. PMID:27904341

Electrophysiologic changes following treatment with organophosphorus-induced delayed neuropathy-producing agents in the adult hen.

PubMed

Robertson, D G; Schwab, B W; Sills, R D; Richardson, R J; Anderson, R J

1987-03-15

Although clinical, pathological, and biochemical effects of organophosphorus-induced delayed neuropathy (OPIDN) have been intensively investigated in the adult hen, detailed electrophysiological studies are lacking. Adult white leghorn hens were treated with a single oral dose of either 30 mg/kg tri-2-cresyl phosphate (TOCP), 750 mg/kg TOCP, 4 mg/kg di-n-butyl-2,2-dichlorovinyl phosphate (DBCV), or 30 mg/kg di-n-butyl-2,2-dichlorovinyl phosphinate (DBCV-P). The 750 mg/kg TOCP and DBCV, but not the 30 mg/kg TOCP and DBCV-P, treatments resulted in clinical signs of OPIDN and mild to marked damage of the tibial nerve 21 days after dose. Twenty-four hr lymphocyte neurotoxic esterase (NTE) inhibition was used as an index of brain NTE inhibition for the various organophosphorus compound (OP) treatment. Twenty-four hr lymphocyte NTE inhibition for 30 mg/kg TOCP, 750 mg/kg TOCP, DBCV, and DBCV-P was 54.1, 87.1, 84.8, and 68.3%, respectively. Twenty-one days after dose, the TOCP-treated hens exhibited some abnormalities in conduction velocity and action potential duration in the tibial or sciatic nerves. No abnormalities were observed in action potential parameters of either the DBCV or DBCV-P treatments. Neurotoxic OP (TOCP and DBCV) treatment resulted in decreased refractoriness in the tibial nerve, increased refractoriness in the sciatic nerve, and elevated strength duration threshold for both nerves. These changes were not present in nerves from DBCV-P (a non-neurotoxic NTE inhibitor)-treated hens. These results suggest that refractory period and strength duration abnormalities in peripheral nerve correlate well with the production of OPIDN and are evident without coincident clinical signs or histopathology.

Determination of some organophosphorus pesticides in water and watermelon samples by microextraction prior to high-performance liquid chromatography.

PubMed

Wang, Chun; Wu, Qiuhua; Wu, Chunxia; Wang, Zhi

2011-11-01

A novel method based on simultaneous liquid-liquid microextraction and carbon nanotube reinforced hollow fiber microporous membrane solid-liquid phase microextraction has been developed for the determination of six organophosphorus pesticides, i.e. isocarbophos, phosmet, parathion-methyl, triazophos, fonofos and phoxim, in water and watermelon samples prior to high-performance liquid chromatography (HPLC). Under the optimum conditions, the method shows a good linearity within a range of 1-200 ng/mL for water samples and 5-200 ng/g for watermelon samples, with the correlation coefficients (r) varying from 0.9990 to 0.9997 and 0.9986 to 0.9995, respectively. The limits of detection (LODs) were in the range between 0.1 and 0.3 ng/mL for water samples and between 1.0 and 1.5 ng/g for watermelon samples. The recoveries of the method at spiking levels of 5.0 and 50.0 ng/mL for water samples were between 85.4 and 100.8%, and at spiking levels of 5.0 and 50.0 ng/g for watermelon samples, they were between 82.6 and 92.4%, with the relative standard deviations (RSDs) varying from 4.5-6.9% and 5.2-7.4%, respectively. The results suggested that the developed method represents a simple, low-cost, high analytes preconcentration and excellent sample cleanup procedure for the determination of organophosphorus pesticides in water and watermelon samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solid phase microextraction applied to the analysis of organophosphorus insecticides in fruits.

PubMed

Fytianos, K; Raikos, N; Theodoridis, G; Velinova, Z; Tsoukali, H

2006-12-01

Trace amounts of organophosphorus pesticides (OPs) were determined in various fruits by headspace solid phase microextraction (HS-SPME) and gas chromatography-nitrogen phosphorous detection (GC-NPD). Sampling from the headspace enhanced method selectivity, whereas at the same time improved fiber life time and method sensitivity. Diazinon, parathion, methyl parathion, malathion and fenithrothion were determined in various fruits: more than 150 samples of 21 types of fruits were studied. SPME-GC-NPD provided a useful and very efficient analytical tool: method linearity ranged from 1.2 to 700 ng/ml. Limits of detection (LODs) and quantitation (LOQs) ranged from 0.03 to 3 ng/ml and 0.12 to 10 ng/ml respectively, values well below the residue limits set by the EU. Less than 2% of the samples were found positive containing amounts higher than the EU limits. The effect of fruit peeling and washing was also investigated.

The relationship between the chemical structure and neurotoxicity of alkyl organophosphorus compounds

PubMed Central

Davies, D. R.; Holland, P.; Rumens, M. J.

1960-01-01

Thirty-six alkyl organophosphorus compounds have been tested for neurotoxicity in the chicken. The individual compounds were chosen to enable the importance of each portion of the molecule to be assessed in relation to the property of neurotoxicity. Seventeen substances were found to be neurotoxic, fifteen for the first time. All of these contained fluorine. On the basis of the results reported, certain predictions have been made about the chemical structure of compounds which would be expected to be neurotoxic. The importance of fluorine suggests that it plays a direct role in the development of the biochemical lesion, and this may occur as the result of its being carried by the molecule as a whole to specific areas in the nervous system. By the action of cholinesterase, the P-F bond may be ruptured and ionic fluorine liberated where it blocks some metabolic cycle. PMID:13814387

The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life

PubMed Central

Atkinson, Gemma C.; Tenson, Tanel; Hauryliuk, Vasili

2011-01-01

RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the “stringent” response and regulator of cellular metabolism. The classical “long” RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the “stringent response” to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual

Luminescence recognition of different organophosphorus pesticides by the luminescent Eu(III)-pyridine-2,6-dicarboxylic acid probe.

PubMed

Azab, Hassan A; Duerkop, Axel; Anwar, Z M; Hussein, Belal H M; Rizk, Moustafa A; Amin, Tarek

2013-01-08

Luminescence quenching of a novel long lived Eu(III)-pyridine-2,6-dicarboxylic acid probe of 1:2 stoichiometric ratio has been studied in 0.10 volume fraction ethanol-water mixture at pH 7.5 (HEPES buffer) in the presence of the organophosphorus pesticides chlorfenvinphos (P1), malathion (P2), azinphos (P3), and paraxon ethyl (P4). The luminescence intensity of Eu(III)-(PDCA)(2) probe decreases as the concentration of the pesticide increases. It was observed that the quenching due to P3 and P4 proceeds via both diffusional and static quenching processes. Direct methods for the determination of the pesticides under investigation have been developed using the luminescence quenching of Eu(III)-pyridine-2,6-dicarboxylic acid probe in solution. The linear range for determination of the selected pesticides is 1.0-35.0 μM. The detection limits were 0.24-0.55 μM for P3, P4, and P1 and 2.5 μM for P2, respectively. The binding constants (K), and thermodynamic parameters of the OPs with Eu(III)-(PDCA)(2) were evaluated. Positive and negative values of entropy (ΔS) and enthalpy (ΔH) changes for Eu(III)-(PDCA)(2)-P1 ternary complex were calculated. As the waters in this study do not contain the above mentioned OPs over the limit detectable by the method, a recovery study was carried out after the addition of the adequate amounts of the organophosphorus pesticides under investigation. Copyright © 2012 Elsevier B.V. All rights reserved.

Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes.

PubMed

Amrein, Beat A; Bauer, Paul; Duarte, Fernanda; Janfalk Carlsson, Åsa; Naworyta, Agata; Mowbray, Sherry L; Widersten, Mikael; Kamerlin, Shina C L

2015-10-02

Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broad range of substrates. The enzyme can be engineered to increase the yield of optically pure products as a result of changes in both enantio- and regioselectivity. It is thus highly attractive in biocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals. The present work aims to establish the principles underlying the activity and selectivity of the enzyme through a combined computational, structural, and kinetic study using the substrate trans -stilbene oxide as a model system. Extensive empirical valence bond simulations have been performed on the wild-type enzyme together with several experimentally characterized mutants. We are able to computationally reproduce the differences between the activities of different stereoisomers of the substrate and the effects of mutations of several active-site residues. In addition, our results indicate the involvement of a previously neglected residue, H104, which is electrostatically linked to the general base H300. We find that this residue, which is highly conserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonated form in order to provide charge balance in an otherwise negatively charged active site. Our data show that unless the active-site charge balance is correctly treated in simulations, it is not possible to generate a physically meaningful model for the enzyme that can accurately reproduce activity and selectivity trends. We also expand our understanding of other catalytic residues, demonstrating in particular the role of a noncanonical residue, E35, as a "backup base" in the absence of H300. Our results provide a detailed view of the main factors driving catalysis and regioselectivity in this enzyme and identify targets for subsequent enzyme design efforts.

Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase

PubMed Central

M., Naresh Kumar; V.B.S.C., Thunuguntla; G.K., Veeramachaneni; B., Chandra Sekhar; Guntupalli, Swapna; J.S., Bondili

2016-01-01

Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G205XS207XG209 and H120XXXXD125. Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser207 of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser207 and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase. PMID:27247428

Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase.

PubMed

M, Naresh Kumar; V B S C, Thunuguntla; G K, Veeramachaneni; B, Chandra Sekhar; Guntupalli, Swapna; J S, Bondili

2016-08-01

Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G(205)XS(207)XG(209) and H(120)XXXXD(125) Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser(207) of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser(207) and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase. © 2016 The Author(s).

Muscarinic receptors mediate the endocrine-disrupting effects of an organophosphorus insecticide in zebrafish.

PubMed

Santos da Rosa, João Gabriel; Alcântara Barcellos, Heloísa Helena de; Fagundes, Michele; Variani, Cristiane; Rossini, Mainara; Kalichak, Fabiana; Koakoski, Gessi; Acosta Oliveira, Thiago; Idalencio, Renan; Frandoloso, Rafael; Piato, Angelo L; José Gil Barcellos, Leonardo

2017-07-01

The glucocorticoid cortisol, the end product of hypothalamus-pituitary-interrenal axis in zebrafish (Danio rerio), is synthesized via steroidogenesis and promotes important physiological regulations in response to a stressor. The failure of this axis leads to inability to cope with environmental challenges preventing adaptive processes in order to restore homeostasis. Pesticides and agrichemicals are widely used, and may constitute an important class of environmental pollutants when reach aquatic ecosystems and nontarget species. These chemical compounds may disrupt hypothalamus-pituitary-interrenal axis by altering synthesis, structure or function of its constituents. We present evidence that organophosphorus exposure disrupts stress response by altering the expression of key genes of the neural steroidogenesis, causing downregulation of star, hsp70, and pomc genes. This appears to be mediated via muscarinic receptors, since the muscarinic antagonist scopolamine blocked these effects. © 2017 Wiley Periodicals, Inc.

Stabilization of an α/β-hydrolase by introducing proline residues: salicylic binding protein 2 from tobacco

PubMed Central

Huang, Jun; Jones, Bryan J.; Kazlauskas, Romas J.

2015-01-01

α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. As a model α/β-hydrolase, we investigated a plant esterase, salicylic acid binding protein 2 (SABP2). SABP2 shows typical stability to urea (unfolding free energy 6.9±1.5 kcal/mol) and to heat inactivation (T1/215 min 49.2±0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homolog or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1) and E215P (+0.9). Introducing proline in the cap domain did not (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/215 min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P-S70P ΔT1/215 min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases. PMID:26110207

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

PubMed Central

Vergnano, Marta; Wan, Chris

2017-01-01

ABSTRACT We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. PMID:28743817

Biocatalytic nerve agent detoxification in fire fighting foams.

PubMed

LeJeune, K E; Russell, A J

1999-03-20

Current events across the globe necessitate rapid technological advances to combat the epidemic of nerve agent chemical weapons. Biocatalysis has emerged as a viable tool in the detoxification of organophosphorus neurotoxins, such as the chemical weapons VX and sarin. Efficient detoxification of contaminated equipment, machinery, and soils are of principal concern. This study describes the incorporation of a biocatalyst (organophosphorus hydrolase, E.C. 3.1.8.1) into conventional formulations of fire fighting foam. The capacity of fire fighting foams to decrease volatilization of contained contaminants, increase surface wettability, and control the rate of enzyme delivery to large areas makes them useful vehicles for enzyme application at surfaces. The performance of enzyme containing foams has been shown to be not only reproducible but also predictable. An empirical model provides reasonable estimations for the amounts of achievable surface decontamination as a function of the important parameters of the system. Theoretical modeling illustrates that the enzyme-containing foam is capable of extracting agent from the surface and is catalytically active at the foam-surface interface and throughout the foam itself. Biocatalytic foam has proven to be an effective, "environmentally friendly" means of surface and soil decontamination.

[High throuput analysis of organophosphorus pesticide residues and their metabolites in animal original foods by dual gas chromatography-dual pulse flame photometric detection].

PubMed

Yang, Lixin; Li, Heli; Miao, Hong; Zeng, Fangang; Li, Ruifeng; Chen, Huijing; Zhao, Yunfeng; Wu, Yongning

2011-10-01

A method was established for the quantitative determination of 54 organophosphorus pesticide residues and their metabolites in foods of animal origin by dual gas chromatography-dual pulse flame photometric detection. Homogenized samples were extracted with acetone and methylene chloride, and cleaned-up by gel permeation chromatography (GPC). The response of each analyte showed a good linearity with a correlation coefficient not less than 0. 99. The recovery experiments were performed by a blank sample spiked at low, medium and high fortification levels. The recoveries for beef, mutton, pork, chicken were in the range of 50. 5% -128. 1% with the relative standard deviations (n = 6) of 1. 1% -25. 5%, which demonstrated the good precision and accuracy of the present method. The limits of detection for the analytes were in the range of 0. 001 -0. 170 mg/kg, and the limits of quantification were in the range of 0. 002 -0. 455 mg/kg. Animal food samples collected from markets such as meat, liver and kidney were analyzed, and the residues of dichlorovos and disulfoton-sulfoxide were found in the some samples. The established method is sensitive and selective enough to detect organophosphorus pesticide residues in animal foods.

Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation

USDA-ARS?s Scientific Manuscript database

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

Structural insights into human microsomal epoxide hydrolase by combined homology modeling, molecular dynamics simulations, and molecular docking calculations.

PubMed

Saenz-Méndez, Patricia; Katz, Aline; Pérez-Kempner, María Lucía; Ventura, Oscar N; Vázquez, Marta

2017-04-01

A new homology model of human microsomal epoxide hydrolase was derived based on multiple templates. The model obtained was fully evaluated, including MD simulations and ensemble-based docking, showing that the quality of the structure is better than that of only previously known model. Particularly, a catalytic triad was clearly identified, in agreement with the experimental information available. Analysis of intermediates in the enzymatic mechanism led to the identification of key residues for substrate binding, stereoselectivity, and intermediate stabilization during the reaction. In particular, we have confirmed the role of the oxyanion hole and the conserved motif (HGXP) in epoxide hydrolases, in excellent agreement with known experimental and computational data on similar systems. The model obtained is the first one that fully agrees with all the experimental observations on the system. Proteins 2017; 85:720-730. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Geotrichum candidum link 1809: hydrolases activity and own method of digestive tract strains biotyping.

PubMed

Kurnatowska, A; Białasiewicz, D

2001-01-01

Hydrolase activity of (enzymograms, biotypes) in Geotrichum candidum, one of the poorly described pathogenic fungi, was studied 81 strains were isolated from oral cavity and faeces of patients with gastrointestinal tract disorders. Axenic strains were differentiated with API 20C Aux and API ZYM tests. Then, enzymograms and biotypes were determined for all strains based on the activity of 19 hydrolases. High variability of enzymograms (17 different types) was found. The highest activity was noted in case of: e2 - alkaline phosphatase, e6 - leucine arylamidase, e11 - acid phosphatase. E5 - lipase, e7 - valine arylamidase, e12 - naphtol-AS-BI-phosphohydrolase and e17 - beta-glucosidase were used for biotyping procedures. Our own system of biotyping of 81 strains of G. candidum was based on the mathematical binominal distribution formula (1 : 4 : 6 : 4 : 1) - all "+"; one "-", three "+"; two "two "+"; three "-", one "+"; all "-". We have found: A (11.1 +/- 3.5%), BI (6.17 +/- 2.67%), B2 (1.23 +/- 1.22%), B4 (4.94 +/- 2.41%), C, (1.23 +/-1.22%), C3 (63.0 +/- 5.4%), D2 (9.88 +/-3.31%), D3 (2.47 +/- 1.72%). Among all strains from 8 various biotypes of G. candidum.

pKa cycling of the general acid/base in glycoside hydrolase families 33 and 34.

PubMed

Yu, Haibo; Griffiths, Thomas M

2014-03-28

Glycoside hydrolase families 33 and 34 catalyse the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates with a net retention of the stereochemistry at the anomeric centre. It is generally believed that the conserved aspartic acid in the active site functions as a general acid to protonate the hydroxyl group of the departing aglycone during glycosylation, and then as a general base to facilitate the nucleophilic attack of the water molecule on the intermediate state during the deglycosylation reaction. The dual role of the general acid/base places specific demands upon its protonation state, and thus pKa values. However, it is not fully understood how this catalytic residue can achieve such pKa cycling during catalysis. We present both MM and combined QM/MM simulations to characterise the pKa values of the proposed catalytic general acid/base in the glycoside hydrolase families 33 and 34. Collectively, our study suggests that the binding of anionic substrates and the local solvation properties along with the neutralisation of the nearby glutamic acid upon glycosylation modulate the electrostatic environment around the general acid/base to achieve its proper protonation states.

An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities.

PubMed

Lee, Yian Hoon; Choo, Candy; Watawana, Mindani I; Jayawardena, Nilakshi; Waisundara, Viduranga Y

2015-11-01

Eighteen edible plants were assessed for their antioxidant potential based on oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, total phenolics, vitamin C content and various lipophilic antioxidants. The inhibitory activities of the plant extracts against the enzymatic activities of α-amylase and α-glucosidase were also evaluated. The antioxidant and starch hydrolase activities of the plants varied widely across a single batch of analysis. The ORAC and DPPH radical scavenging EC50 values varied between 298 and 1984 Trolox equivalents g(-1) fresh weight and between 91 and 533 mg kg(-1) fresh weight, respectively. The total phenolics and vitamin C contents varied between 32 and 125 mg gallic acid equivalents g(-1) fresh weight and between 96 and 285 µg g(-1) fresh weight, respectively. All the plants contained neoxanthin, violaxanthin, and α- and β-carotene in varying amounts. Coccinia grandis, Asparagus racemosus, Costus speciosus, Amaranthus viridis and Annona muricata displayed the highest inhibitory activities against starch hydrolases. They were the most efficient against the breakdown of seven starches exposed to the two enzymes as well. Overall, the edible plants were observed to display a high antioxidant potential with starch hydrolase inhibitory properties, which were beneficial in their being recognized as functional food. © 2014 Society of Chemical Industry.

Recovery of cholinesterase activity in mallard ducklings administered organophosphorus pesticides

USGS Publications Warehouse

Fleming, W.J.; Bradbury, S.P.

1981-01-01

Oral doses of the organophosphorus pesticides acephate, dicrotophos, fensulfothion, fonofos, malathion, and parathion were administered to mallard ducklings (Anas platyrhynchos), and brain and plasma cholinesterase (ChE) activities were determined for up to 77 d after dosing. In vivo recovery of brain ChE activity to within 2 standard deviations of the mean activity of undosed birds occurred within 8 d, after being depressed an average of 25-58% at 24 h after dosing. In vivo recovery of plasma ChE appeared as fast as or faster than that of brain, but the pattern of recovery was more erratic and therefore statistical comparison with brain ChE recovery was not attempted. In vitro tests indicated that the potential for dephosphorylation to contribute to in vivo recovery of inhibited brain ChE differed among chemical treatments. Some ducklings died as a result of organophosphate dosing. In an experiment in which ducklings within each treatment group received the same dose (mg/kg), the brain ChE activity in birds that died was less than that in birds that survived. Brain ChE activities in ducklings that died were significantly different among pesticide treatments: fensulfothion > parathion> acephate > malathion (p < 0.05).

Neurotoxicity in Preclinical Models of Occupational Exposure to Organophosphorus Compounds.

PubMed

Voorhees, Jaymie R; Rohlman, Diane S; Lein, Pamela J; Pieper, Andrew A

2016-01-01

Organophosphorus (OPs) compounds are widely used as insecticides, plasticizers, and fuel additives. These compounds potently inhibit acetylcholinesterase (AChE), the enzyme that inactivates acetylcholine at neuronal synapses, and acute exposure to high OP levels can cause cholinergic crisis in humans and animals. Evidence further suggests that repeated exposure to lower OP levels insufficient to cause cholinergic crisis, frequently encountered in the occupational setting, also pose serious risks to people. For example, multiple epidemiological studies have identified associations between occupational OP exposure and neurodegenerative disease, psychiatric illness, and sensorimotor deficits. Rigorous scientific investigation of the basic science mechanisms underlying these epidemiological findings requires valid preclinical models in which tightly-regulated exposure paradigms can be correlated with neurotoxicity. Here, we review the experimental models of occupational OP exposure currently used in the field. We found that animal studies simulating occupational OP exposures do indeed show evidence of neurotoxicity, and that utilization of these models is helping illuminate the mechanisms underlying OP-induced neurological sequelae. Still, further work is necessary to evaluate exposure levels, protection methods, and treatment strategies, which taken together could serve to modify guidelines for improving workplace conditions globally.

Neurotoxicity in Preclinical Models of Occupational Exposure to Organophosphorus Compounds

PubMed Central

Voorhees, Jaymie R.; Rohlman, Diane S.; Lein, Pamela J.; Pieper, Andrew A.

2017-01-01

Organophosphorus (OPs) compounds are widely used as insecticides, plasticizers, and fuel additives. These compounds potently inhibit acetylcholinesterase (AChE), the enzyme that inactivates acetylcholine at neuronal synapses, and acute exposure to high OP levels can cause cholinergic crisis in humans and animals. Evidence further suggests that repeated exposure to lower OP levels insufficient to cause cholinergic crisis, frequently encountered in the occupational setting, also pose serious risks to people. For example, multiple epidemiological studies have identified associations between occupational OP exposure and neurodegenerative disease, psychiatric illness, and sensorimotor deficits. Rigorous scientific investigation of the basic science mechanisms underlying these epidemiological findings requires valid preclinical models in which tightly-regulated exposure paradigms can be correlated with neurotoxicity. Here, we review the experimental models of occupational OP exposure currently used in the field. We found that animal studies simulating occupational OP exposures do indeed show evidence of neurotoxicity, and that utilization of these models is helping illuminate the mechanisms underlying OP-induced neurological sequelae. Still, further work is necessary to evaluate exposure levels, protection methods, and treatment strategies, which taken together could serve to modify guidelines for improving workplace conditions globally. PMID:28149268

Cresyl Saligenin Phosphate, an Organophosphorus Toxicant, Makes Covalent Adducts with Histidine, Lysine and Tyrosine Residues of Human Serum Albumin

PubMed Central

Liyasova, Mariya S.; Schopfer, Lawrence M.; Lockridge, Oksana

2012-01-01

CBDP (2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide) is a toxic organophosphorus compound. It is generated in vivo from tri-ortho-cresyl phosphate (TOCP), a component of jet engine oil and hydraulic fluids. Exposure to TOCP was proven to occur on board aircraft by finding CBDP-derived phospho-butyrylcholinesterase in the blood of passengers. Adducts on BChE however do not explain the toxicity of CBDP. Critical target proteins of CBDP are yet to be identified. Our goal was to facilitate the search for the critical targets of CBDP by determining the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human albumin as a model protein. Mass spectral analysis of the tryptic digest of CBDP-treated human albumin revealed adducts on His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, Lys-525. Adducts formed on tyrosine residues were different from those formed on histidines and lysines. Tyrosines were organophosphorylated by CBDP, while histidine and lysine residues were alkylated. This is the first report of an organophosphorus compound with both phosphorylating and alkylating properties. The hydroxybenzyl adduct on histidine is novel. The ability of CBDP to form stable adducts on histidine, tyrosine and lysine allows one to consider new mechanisms of toxicity from TOCP exposure. PMID:22793878

Cresyl saligenin phosphate, an organophosphorus toxicant, makes covalent adducts with histidine, lysine, and tyrosine residues of human serum albumin.

PubMed

Liyasova, Mariya S; Schopfer, Lawrence M; Lockridge, Oksana

2012-08-20

CBDP [2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide] is a toxic organophosphorus compound. It is generated in vivo from tri-ortho-cresyl phosphate (TOCP), a component of jet engine oil and hydraulic fluids. Exposure to TOCP was proven to occur on board aircraft by finding CBDP-derived phospho-butyrylcholinesterase in the blood of passengers. Adducts on BChE, however, do not explain the toxicity of CBDP. Critical target proteins of CBDP are yet to be identified. Our goal was to facilitate the search for the critical targets of CBDP by determining the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human albumin as a model protein. Mass spectral analysis of the tryptic digest of CBDP-treated human albumin revealed adducts on His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, and Lys-525. Adducts formed on tyrosine residues were different from those formed on histidines and lysines. Tyrosines were organophosphorylated by CBDP, while histidine and lysine residues were alkylated. This is the first report of an organophosphorus compound with both phosphorylating and alkylating properties. The o-hydroxybenzyl adduct on histidine is novel. The ability of CBDP to form stable adducts on histidine, tyrosine, and lysine allows one to consider new mechanisms of toxicity from TOCP exposure.

In Vivo Microdialysis and Electroencephalographic Activity in Freely Moving Guinea Pigs Exposed to Organophosphorus Nerve Agents Sarin and VX: Analysis of Acetylcholine and Glutamate

DTIC Science & Technology

2011-01-01

3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE In vivo microdialysis and electroencephalographic activity in freely moving guinea pigs 5a...microdialysis and electroencephalographic activity in freely moving guinea pigs exposed to organophosphorus nerve agents sarin and VX: analysis of...brain seizure activity . This robust double multi- variate design provides greater fidelity when comparing data while also reducing the required number

[Semicontinuous cultivation of fungi of the genus Aspergillus, producers of hydrolases].

PubMed

Blieva, R K

1982-01-01

The production of exohydrolases (alpha-amylase and pectinase) by fungi belonging to the genus Aspergillus was studied in the course of batch cultivation and, if immobilized cells were used, in the semicontinuous regime of growth. The cells were immobilized on a fixed filtering plate and on floating, in the growth medium, polyhedrons. Such a cultivation of immobilized microbial cells in the semicontinuous regime of growth on submerged polyhedrons freely floating in the nutrient medium makes it possible to cultivate the cells for 1.5 months with the active production of exocellular hydrolases. Under these conditions, Aspergillus oryzae 3-9-15 produces more alpha-amylase and A. awamori synthesizes more pectinases.

Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and neurotoxicity.

PubMed

Behl, Mamta; Hsieh, Jui-Hua; Shafer, Timothy J; Mundy, William R; Rice, Julie R; Boyd, Windy A; Freedman, Jonathan H; Hunter, E Sidney; Jarema, Kimberly A; Padilla, Stephanie; Tice, Raymond R

2015-01-01

Due to their toxicity and persistence in the environment, brominated flame retardants (BFRs) are being phased out of commercial use, leading to the increased use of alternative chemicals such as the organophosphorus flame retardants (OPFRs). There is, however, limited information on the potential health effects of OPFRs. Due to the structural similarity of the OPFRs to organophosphorus insecticides, there is concern regarding developmental toxicity and neurotoxicity. In response, we evaluated a set of OPFRs (triphenyl phosphate [TPHP]), isopropylated phenyl phosphate [IPP], 2-ethylhexyl diphenyl phosphate [EHDP], tert-butylated phenyl diphenyl phosphate [BPDP], trimethyl phenyl phosphate [TMPP], isodecyl diphenyl phosphate [IDDP], (tris(1,3-dichloroisopropyl) phosphate [TDCIPP], and tris(2-chloroethyl)phosphate [TCEP]) in a battery of cell-based in vitro assays and alternative model organisms and compared the results to those obtained for two classical BFRs (3,3',5,5'-tetrabromobisphenol A [TBBPA] and 2,2'4,4'-brominated diphenyl ether [BDE-47]). The assays used evaluated the effects of chemicals on the differentiation of mouse embryonic stem cells, the proliferation and growth of human neural stem cells, rat neuronal growth and network activity, and development of nematode (Caenorhabditis elegans) and zebrafish (Danio rerio). All assays were performed in a concentration-response format, allowing for the determination of the point of departure (POD: the lowest concentration where a chemically-induced response exceeds background noise). The majority of OPFRs (8/9) were active in multiple assays in the range of 1-10 μM, most of which had comparable activity to the BFRs TBBPA and BDE-47. TCEP was negative in all assays. The results indicate that the replacement OPFRs, with the exception of TCEP, showed comparable activity to the two BFRs in the assays tested. Based on these results, more comprehensive studies are warranted to further characterize the potential hazard

Genetic basis for the resistance of Staphylococcus aureus to peptidoglycan hydrolase by comparative transcriptome and whole genome sequence analysis

USDA-ARS?s Scientific Manuscript database

Background: Lysostaphin is a glycyl-glycine bacteriocin peptidoglycan hydrolase secreted by Staphylococcus simulans for degrading the peptidoglycan moieties in Staphylococcus aureus cell walls which result in cell lysis. There are known mechanisms of resistance to lysostaphin, e.g. serine in place...

Non-muscarinic therapeutic targets for acute organophosphorus poisoning.

PubMed

Rosenbaum, Christopher; Bird, Steven B

2010-12-01

Organophosphorus (OP) pesticides are a broad class of acetylcholinesterase inhibitors that are responsible for tremendous morbidity and mortality worldwide, contributing to an estimated 300,000 deaths annually. Current pharmacotherapy for acute OP poisoning includes the use of atropine, an oxime, and benzodiazepines. However, even with such therapy, the mortality from these agents is as high as 40%. It is increasingly recognized that not all OPs are the same. Significant differences exist in their toxicity, lipophilicity, and response to oxime therapy. Other non-muscarinic effects of OP pesticides exist, such as acute and chronic neuromuscular junction failure and central respiratory failure. In part because most of the mortality from these chemicals takes place in the developing world, little National Institutes of Health (NIH) research has been directed towards these agents. However, the similar mechanism of action of OP pesticides and the military nerve agents, along with increasing concerns about chemical terrorism has lead to the formation of the NIH Countermeasures Against Chemical Threats (CounterACT) Program. As part of the CounterACT Program, the NIH has recently designated six OP pesticides as "threat agents". This concept paper describes some of the knowledge gaps related to non-muscarinic effects of OP pesticides and highlights needed areas of further research. Leveraging the current NIH interest in these chemicals to medical necessities in the developing world offers the possibility of delivering new therapeutics where they are needed on a daily basis.

Subchronic organophosphorus ester-induced delayed neurotoxicity in mallards

USGS Publications Warehouse

Hoffman, D.J.; Sileo, L.; Murray, H.C.

1984-01-01

Eighteen-week-old mallard hens received 0, 10, 30, 90, or 270 ppm technical grade EPN (phenylphosphonothioic acid O-ethyl-O-4-nitrophenyl ester) in the diet for 90 days. Ataxia was first observed in the 270-ppm group after 16 days, in the 90-ppm group after 20 days, in the 30-ppm group after 38 days; 10 ppm failed to produce ataxia. By the end of 90 days all 6 birds in the 270-ppm group exhibited ataxia or paralysis whereas 5 of 6 birds in the 90-ppm group and 2 of 6 birds in the 30-ppm group were visibly affected. Treatment with 30 ppm or more resulted in a significant reduction in body weight. Brain neurotoxic esterase activity was inhibited by averages of 16, 69, 73, and 74% in the 10-, 30-, 90-, and 270-ppm groups, respectively. Brain acetylcholinesterase, plasma cholinesterase, and plasma alkaline phosphatase were significantly inhibited as well. Distinct histopathological effects were seen in the 30-, 90-, and 270-ppm groups which included demyelination and degeneration of axons of the spinal cord. Additional ducks were exposed in a similar manner to 60-, 270-, or 540-ppm leptophos (phosphonothioic acid O-4-bromo-2,5-dichlorophenyl-O-methylphenyl ester) which resulted in similar behavioral, biochemical, and histopathological alterations. These findings indicate that adult mallards are probably somewhat less sensitive than chickens to subchronic dietary exposure to organophosphorus insecticides that induce delayed neurotoxicity.

Localization of cholinesterases in the chicken nervous system and the problem of the selective neurotoxicity of organophosphorus compounds

PubMed Central

Cavanagh, J. B.; Holland, P.

1961-01-01

Using the thiocholine method, a restricted survey has been made of cholinesterases in the spinal cord and brain stem of the chicken. No simple relation between sites of selective damage in organophosphorus neurotoxicity and centres of cholinesterase activity could be adduced. Moreover, no significant differences between species susceptible and insusceptible to poisoning by these compounds were found by this method. It is concluded that, while cholinesterase may well play an intermediary role in the intoxication, other factors determine the selective damage to certain neurones and their processes. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6 PMID:13691740

Structure-activity correlations for organophosphorus ester anticholinesterases. Part 2: CNDO/2 calculations applied to ester hydrolysis rates

NASA Technical Reports Server (NTRS)

Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

1984-01-01

Quantitative structure-activity relationships are presented for the hydrolysis of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. CNDO/2 calculations provide values for molecular parameters that correlate with alkaline hydrolysis rates. For each subset of esters with the same leaving group, X, the CNDO-derived net atomic charge at the central phosphorus atom correlates well with the alkaline hydrolysis rate constants. For the whole set of esters with different leaving groups, equations are derived that relate charge, orbital energy and bond order to the hydrolysis rate constants.

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

PubMed

Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

2016-11-15

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.

PubMed

Simon, Gabriel M; Cravatt, Benjamin F

2010-04-09

Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.

3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

EPA Science Inventory

Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylu...

Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

PubMed

Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

2016-06-01

Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.

PubMed

Chae, J P; Valeriano, V D; Kim, G-B; Kang, D-K

2013-01-01

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l(-1) isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni(2+) -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes. © 2012 The Society for Applied Microbiology.

Microsomal epoxide hydrolase of rat liver. Purification and characterization of enzyme fractions with different chromatographic characteristics.

PubMed Central

Bulleid, N J; Graham, A B; Craft, J A

1986-01-01

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms. Images Fig. 2. Fig. 3. PMID:3082328

Serum concentration of ubiquitin c-terminal hydrolase-L1 in detecting severity of traumatic brain injury

NASA Astrophysics Data System (ADS)

Siahaan, A. M. P.; Japardi, I.; Hakim, A. A.

2018-03-01

One of the main problems with ahead injury is assessing the severity. While physical examination and imaging had limitations, neuronal damage markers, ubiquitin C-terminal hydrolase-L1 (UCH-L1), released in theblood may provide valuable information about diagnosis the traumatic brain injury (TBI).Analyzing the concentrations of serum ubiquitin C-terminal hydrolase-L1 (UCH-L1), there must have a neuronal injury biomarker, in theTBI patients serum and their association with clinical characteristics and outcome. There were 80 TBI subjects, and there are mild, moderate, and severe involved in this study of case- control. By using ELISA, we studied the profile of serum UCH-L1 levels for TBI patients. TheUCH-L1 serum level of moderate and severe head injury is higher than in mild head injury (p<.001), but we didn’t find aspecific difference between moderate and severe head injury patients. There is no particular correlation found between serum UCH-L1 level and outcome. Serum levels of UCH-L1 appear to have potential clinical utility in diagnosing TBI but do not correlate with outcome.

Simultaneous determination of organophosphorus pesticides and phthalates in baby food samples by ultrasound-vortex-assisted liquid-liquid microextraction and GC-IT/MS.

PubMed

Notardonato, Ivan; Salimei, Elisabetta; Russo, Mario Vincenzo; Avino, Pasquale

2018-05-01

Baby foods are either a soft, liquid paste or an easily chewed food since babies lack developed muscles and teeth to chew effectively. Babies typically move to consuming baby food once nursing or formula is not sufficient for the child's appetite. Some commercial baby foods have been criticized for their contents. This article focuses on the simultaneous determination of organophosphorus pesticides and phthalates by means of a method based on ultrasound-vortex-assisted liquid-liquid microextraction coupled with gas chromatography-ion trap mass spectrometry (GC-IT/MS). The protocol developed allowed the determination of six phthalates [dimethyl phthalate, diethyl phthalate, dibutyl phthalate, isobutyl cyclohexyl phthalate, benzyl butyl phthalate, bis(2-ethylhexyl) phthalate] and 19 organophosphorus pesticides. Freeze-dried product samples (0.1-0.2 g) were dissolved in 10 mL of warm distilled water along with 5 μL of an internal standard (anthracene at 10 mg mL -1 in acetone): the choice of extraction solvent was studied, with the most suitable being n-heptane, which is used for phthalate determination in similar matrices. The solution, held for 5 min in a vortex mixer and for 6 min in a 100-W ultrasonic bath to favor solvent dispersion and consequently analyte extraction, was centrifuged at 4000 rpm for 30 min. Then 1 μL was injected into the GC-IT/MS system (SE-54 capillary column; length 30 m, inner diameter 250 μm, film thickness 0.25 μm). All analytical parameters investigated are discussed in depth. The method was applied to real commercial freeze-dried samples: significant contaminant concentrations were not found. Graphical abstract Simultaneous and sensitive determination of organophosphorus pesticides and phthalates in baby foods by the ultrasound-vortex-assisted liquid-liquid microextraction ֪gas chromatography-ion trap mass spectrometry procedure. 1 methacrifos, 2 pirofos, 3 phorate, 4 seraphos, 5 diazinon, 6 etrimphos, 7 dichlofenthion, 8

PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

PubMed Central

Hugouvieux-Cotte-Pattat, Nicole; Shevchik, Vladimir E.; Nasser, William

2002-01-01

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-α-d-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind

Structural analysis of a glycosides hydrolase family 42 cold-adapted ß-galactosidase from Rahnella sp. R3

USDA-ARS?s Scientific Manuscript database

The ß-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-ß-Gal), exhibits high activity at low temperature. R-ß-Gal is a member of the glycoside hydrolases family 42 (GH42), and forms a 225 kDa trimeric structure in solution. The X-ray crystal structure of R-ß-Gal was determi...

Detection of Organophosphorus Pesticides with Colorimetry and Computer Image Analysis.

PubMed

Li, Yanjie; Hou, Changjun; Lei, Jincan; Deng, Bo; Huang, Jing; Yang, Mei

2016-01-01

Organophosphorus pesticides (OPs) represent a very important class of pesticides that are widely used in agriculture because of their relatively high-performance and moderate environmental persistence, hence the sensitive and specific detection of OPs is highly significant. Based on the inhibitory effect of acetylcholinesterase (AChE) induced by inhibitors, including OPs and carbamates, a colorimetric analysis was used for detection of OPs with computer image analysis of color density in CMYK (cyan, magenta, yellow and black) color space and non-linear modeling. The results showed that there was a gradually weakened trend of yellow intensity with the increase of the concentration of dichlorvos. The quantitative analysis of dichlorvos was achieved by Artificial Neural Network (ANN) modeling, and the results showed that the established model had a good predictive ability between training sets and predictive sets. Real cabbage samples containing dichlorvos were detected by colorimetry and gas chromatography (GC), respectively. The results showed that there was no significant difference between colorimetry and GC (P > 0.05). The experiments of accuracy, precision and repeatability revealed good performance for detection of OPs. AChE can also be inhibited by carbamates, and therefore this method has potential applications in real samples for OPs and carbamates because of high selectivity and sensitivity.

Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents.

PubMed

Albuquerque, Edson X; Pereira, Edna F R; Aracava, Yasco; Fawcett, William P; Oliveira, Maristela; Randall, William R; Hamilton, Tracey A; Kan, Robert K; Romano, James A; Adler, Michael

2006-08-29

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer's disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning.

Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents

PubMed Central

Albuquerque, Edson X.; Pereira, Edna F. R.; Aracava, Yasco; Fawcett, William P.; Oliveira, Maristela; Randall, William R.; Hamilton, Tracey A.; Kan, Robert K.; Romano, James A.; Adler, Michael

2006-01-01

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer’s disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning. PMID:16914529

Lanthanide sorbent based on magnetite nanoparticles functionalized with organophosphorus extractants

PubMed Central

Basualto, Carlos; Gaete, José; Molina, Lorena; Valenzuela, Fernando; Yañez, Claudia; Marco, Jose F

2015-01-01

In this work, an adsorbent was prepared based on the attachment of organophosphorus acid extractants, namely, D2EHPA, CYANEX 272, and CYANEX 301, to the surface of superparamagnetic magnetite (Fe3O4) nanoparticles. The synthesized nanoparticles were coated with oleic acid, first by a chemisorption mechanism and later by the respective extractant via physical adsorption. The obtained core–shell functionalized magnetite nanoparticle composites were characterized by dynamic light scattering, scanning electron microscopy, transmission electron microscopy, thermogravimetry, infrared absorption and vibrating sample magnetometry. All the prepared nanoparticles exhibited a high saturation magnetization capacity that varied between 72 and 46 emu g−1 and decreased as the magnetite nanoparticle was coated with oleic acid and functionalized. The scope of this study also included adsorption tests for lanthanum, cerium, praseodymium, and neodymium and the corresponding analysis of their results. Sorption tests indicated that the functionalized nanoparticles were able to extract the four studied lanthanide metal ions, although the best extraction performance was observed when the sorbent was functionalized with CYANEX 272, which resulted in a loading capacity of approximately 12–14 mgLa/gMNP. The magnetization of the synthesized nanoparticles was verified during the separation of the lanthanide-loaded sorbent from the raffinate by using a conventional magnet. PMID:27877811

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

PubMed Central

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-01-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase. PMID:12429023

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

PubMed

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-03-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

Household organophosphorus pesticide use and Parkinson’s disease

PubMed Central

Narayan, Shilpa; Liew, Zeyan; Paul, Kimberly; Lee, Pei-Chen; Sinsheimer, Janet S; Bronstein, Jeff M; Ritz, Beate

2013-01-01

Background Household pesticide use is widespread in the USA. Since the 1970s, organophosphorus chemicals (OPs) have been common active ingredients in these products. Parkinson’s disease (PD) has been linked to pesticide exposures but little is known about the contributions of chronic exposures to household pesticides. Here we investigate whether long-term use of household pesticides, especially those containing OPs, increases the odds of PD. Methods In a population-based case-control study, we assessed frequency of household pesticide use for 357 cases and 807 controls, relying on the California Department of Pesticide Regulation product label database to identify ingredients in reported household pesticide products and the Pesticide Action Network pesticide database of chemical ingredients. Using logistic regression we estimated the effects of household pesticide use. Results Frequent use of any household pesticide increased the odds of PD by 47% [odds ratio (OR) = 1.47, (95% confidence interval (CI): 1.13, 1.92)]; frequent use of products containing OPs increased the odds of PD more strongly by 71% [OR = 1.71, (95% CI: 1.21, 2.41)] and frequent organothiophosphate use almost doubled the odds of PD. Sensitivity analyses showed that estimated effects were independent of other pesticide exposures (ambient and occupational) and the largest odds ratios were estimated for frequent OP users who were carriers of the 192QQ paraoxonase genetic variant related to slower detoxification of OPs. Conclusions We provide evidence that household use of OP pesticides is associated with an increased risk of developing PD. PMID:24057998

[Comparative analysis of sensitivity of proteases (chymotrypsin and trypsin) and cholinesterases of different origin to some organophosphorus inhibitors].

PubMed

Rozengart, E V

2009-01-01

The antichymotrypsin, antitrypsin, and anticholinesterase efficiencies of four homologous series of organophosphorus inhibitors are compared: O-ethyl-S-(n-alkyl)methylthiophosphonates, O-(n-alkyl)-S-(n-butyl)methylthiophosphonates, O-(n-alkyl)-S-beta-(ethylmercaptoethylene)methylthiophosphonates, and their methylsulfomethylates. As sources of a-chymotrypsin and trypsin, commercial compounds of Worthington Biochemical Corporation and Leningrad Myasokombinat were tested. Bimolecular constant of the reaction rate was used as the measure of antienzyme efficiency. In all cases, the antichymotrypsin efficiency was lower, while the antitrypsin--essentially higher than the anticholinesterase activity of the studied inhibitors. These differences were found to much depend both on the inhibitor structure and on nature of the cholinesterase compounds.

Consumption of fruits and vegetables and probabilistic assessment of the cumulative acute exposure to organophosphorus and carbamate pesticides of schoolchildren in Slovenia.

PubMed

Blaznik, Urška; Yngve, Agneta; Eržen, Ivan; Hlastan Ribič, Cirila

2016-02-01

Adequate consumption of fruits and vegetables is a part of recommendations for a healthy diet. The aim of the present study was to assess acute cumulative dietary exposure to organophosphorus and carbamate pesticides via fruit and vegetable consumption by the population of schoolchildren aged 11-12 years and the level of risk for their health. Cumulative probabilistic risk assessment methodology with the index compound approach was applied. Slovenia, primary schools. Schoolchildren (n 1145) from thirty-one primary schools in Slovenia. Children were part of the PRO GREENS study 2009/10 which assessed 11-year-olds' consumption of fruit and vegetables in ten European countries. The cumulative acute exposure amounted to 8.3 (95% CI 7.7, 10.6) % of the acute reference dose (ARfD) for acephate as index compound (100 µg/kg body weight per d) at the 99.9th percentile for daily intake and to 4.5 (95% CI 3.5, 4.7) % of the ARfD at the 99.9th percentile for intakes during school time and at lunch. Apples, bananas, oranges and lettuce contributed most to the total acute pesticides intake. The estimations showed that acute dietary exposure to organophosphorus and carbamate pesticides is not a health concern for schoolchildren with the assessed dietary patterns of fruit and vegetable consumption.

Biosensors and their applications in detection of organophosphorus pesticides in the environment.

PubMed

Hassani, Shokoufeh; Momtaz, Saeideh; Vakhshiteh, Faezeh; Maghsoudi, Armin Salek; Ganjali, Mohammad Reza; Norouzi, Parviz; Abdollahi, Mohammad

2017-01-01

This review discusses the past and recent advancements of biosensors focusing on detection of organophosphorus pesticides (OPs) due to their exceptional use during the last decades. Apart from agricultural benefits, OPs also impose adverse toxicological effects on animal and human population. Conventional approaches such as chromatographic techniques used for pesticide detection are associated with several limitations. A biosensor technology is unique due to the detection sensitivity, selectivity, remarkable performance capabilities, simplicity and on-site operation, fabrication and incorporation with nanomaterials. This study also provided specifications of the most OPs biosensors reported until today based on their transducer system. In addition, we highlighted the application of advanced complementary materials and analysis techniques in OPs detection systems. The availability of these new materials associated with new sensing techniques has led to introduction of easy-to-use analytical tools of high sensitivity and specificity in the design and construction of OPs biosensors. In this review, we elaborated the achievements in sensing systems concerning innovative nanomaterials and analytical techniques with emphasis on OPs.

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos

PubMed Central

Kim, Young Ah; Lee, Eun-Hye; Kim, Kwang-Ok; Lee, Yong Tae; Hammock, Bruce D.; Lee, Hye-Sung

2014-01-01

An immunochromatographic assay (ICA) based on competitive antigen-coated format using colloidal gold as the label was developed for the detection of the organophosphorus insecticide chlorpyrifos. The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with chlorpyrifos hapten-OVA conjugate (test line) and anti-mouse IgG (control line). Based on the fact that the competition is between the migrating analyte in the sample and the analyte hapten immobilized on the test strip for the binding sites of the antibody-colloidal gold (Ab-CG) conjugate migrating on the test strip, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. This criterion was utilized for the confirmation of appropriateness of a nitrocellulose (NC) membrane for chlorpyrifos ICA. The detection limit of the ICA for chlorpyrifos standard and chlorpyrifos spiked into agricultural samples were 10 and 50 ng mL−1, respectively. The assay time for the ICA test was less than 10 min, suitable for rapid on-site testing of chlorpyrifos. PMID:21504817

Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

USDA-ARS?s Scientific Manuscript database

Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

PubMed Central

Attai, Hedieh; Rimbey, Jeanette; Smith, George P.

2017-01-01

ABSTRACT To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens. Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens. The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative phage peptidoglycan hydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N-acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens, may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The

Identification of N-acylethanolamines in Dictyostelium discoideum and confirmation of their hydrolysis by fatty acid amide hydrolase[S

PubMed Central

Hayes, Alexander C.; Stupak, Jacek; Li, Jianjun; Cox, Andrew D.

2013-01-01

N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotypes. This has led to the development of pharmaceutical-based therapies for a variety of conditions targeting FAAH. Recently, our group identified a functional FAAH homolog in Dictyostelium discoideum, leading to our hypothesis that D. discoideum also possesses NAEs. In this study, we provide a further characterization of FAAH and identify NAEs in D. discoideum for the first time. We also demonstrate the ability to modulate their levels in vivo through the use of a semispecific FAAH inhibitor and confirm that these NAEs are FAAH substrates through in vitro studies. We believe the demonstration of the in vivo modulation of NAE levels suggests that D. discoideum could be a good simple model organism in which to study NAE-mediated signaling. PMID:23187822

Novel propanamides as fatty acid amide hydrolase inhibitors.

PubMed

Deplano, Alessandro; Morgillo, Carmine Marco; Demurtas, Monica; Björklund, Emmelie; Cipriano, Mariateresa; Svensson, Mona; Hashemian, Sanaz; Smaldone, Giovanni; Pedone, Emilia; Luque, F Javier; Cabiddu, Maria G; Novellino, Ettore; Fowler, Christopher J; Catalanotti, Bruno; Onnis, Valentina

2017-08-18

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

USDA-ARS?s Scientific Manuscript database

A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

PubMed Central

Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

2014-01-01

Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

Identification of the sequence motif of glycoside hydrolase 13 family members

PubMed Central

Kumar, Vikash

2011-01-01

A bioinformatics analysis of sequences of enzymes of the glycoside hydrolase (GH) 13 family members such as α-amylase, cyclodextrin glycosyltransferase (CGTase), branching enzyme and cyclomaltodextrinase has been carried out in order to find out the sequence motifs that govern the reactions specificities of these enzymes by using hidden Markov model (HMM) profile. This analysis suggests the existence of such sequence motifs and residues of these motifs constituting the −1 to +3 catalytic subsites of the enzyme. Hence, by introducing mutations in the residues of these four subsites, one can change the reaction specificities of the enzymes. In general it has been observed that α -amylase sequence motif have low sequence conservation than rest of the motifs of the GH13 family members. PMID:21544166

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar tomore » tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.« less

Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

PubMed

Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

2009-10-26

The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

PubMed

Brulc, Jennifer M; Antonopoulos, Dionysios A; Miller, Margret E Berg; Wilson, Melissa K; Yannarell, Anthony C; Dinsdale, Elizabeth A; Edwards, Robert E; Frank, Edward D; Emerson, Joanne B; Wacklin, Pirjo; Coutinho, Pedro M; Henrissat, Bernard; Nelson, Karen E; White, Bryan A

2009-02-10

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

Rapid kinetic characterization of glycosyl hydrolases based on oxime derivatization and nanostructure-initiator mass spectrometry (NIMS).

PubMed

Deng, Kai; Takasuka, Taichi E; Heins, Richard; Cheng, Xiaoliang; Bergeman, Lai F; Shi, Jian; Aschenbrener, Ryan; Deutsch, Sam; Singh, Seema; Sale, Kenneth L; Simmons, Blake A; Adams, Paul D; Singh, Anup K; Fox, Brian G; Northen, Trent R

2014-07-18

Glycoside hydrolases (GHs) are critical to cycling of plant biomass in the environment, digestion of complex polysaccharides by the human gut microbiome, and industrial activities such as deployment of cellulosic biofuels. High-throughput sequencing methods show tremendous sequence diversity among GHs, yet relatively few examples from the over 150,000 unique domain arrangements containing GHs have been functionally characterized. Here, we show how cell-free expression, bioconjugate chemistry, and surface-based mass spectrometry can be used to study glycoside hydrolase reactions with plant biomass. Detection of soluble products is achieved by coupling a unique chemical probe to the reducing end of oligosaccharides in a stable oxime linkage, while the use of (13)C-labeled monosaccharide standards (xylose and glucose) allows quantitation of the derivatized glycans. We apply this oxime-based nanostructure-initiator mass spectrometry (NIMS) method to characterize the functional diversity of GHs secreted by Clostridium thermocellum, a model cellulolytic organism. New reaction specificities are identified, and differences in rates and yields of individual enzymes are demonstrated in reactions with biomass substrates. Numerical analyses of time series data suggests that synergistic combinations of mono- and multifunctional GHs can decrease the complexity of enzymes needed for the hydrolysis of plant biomass during the production of biofuels.

Detection of vapor-phase organophosphate threats using wearable conformable integrated epidermal and textile wireless biosensor systems.

PubMed

Mishra, Rupesh K; Martín, Aida; Nakagawa, Tatsuo; Barfidokht, Abbas; Lu, Xialong; Sempionatto, Juliane R; Lyu, Kay Mengjia; Karajic, Aleksandar; Musameh, Mustafa M; Kyratzis, Ilias L; Wang, Joseph

2018-03-15

Flexible epidermal tattoo and textile-based electrochemical biosensors have been developed for vapor-phase detection of organophosphorus (OP) nerve agents. These new wearable sensors, based on stretchable organophosphorus hydrolase (OPH) enzyme electrodes, are coupled with a fully integrated conformal flexible electronic interface that offers rapid and selective square-wave voltammetric detection of OP vapor threats and wireless data transmission to a mobile device. The epidermal tattoo and textile sensors display a good reproducibility (with RSD of 2.5% and 4.2%, respectively), along with good discrimination against potential interferences and linearity over the 90-300mg/L range, with a sensitivity of 10.7µA∙cm 3 ∙mg -1 (R 2 = 0.983) and detection limit of 12mg/L in terms of OP air density. Stress-enduring inks, used for printing the electrode transducers, ensure resilience against mechanical deformations associated with textile and skin-based on-body sensing operations. Theoretical simulations are used to estimate the OP air density over the sensor surface. These fully integrated wearable wireless tattoo and textile-based nerve-agent vapor biosensor systems offer considerable promise for rapid warning regarding personal exposure to OP nerve-agent vapors in variety of decentralized security applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

PubMed

Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

2017-01-01

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases.

PubMed

Janeček, Štefan; Svensson, Birte; MacGregor, E Ann

2014-04-01

α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4-7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

PubMed

Zhi, Tiantian; Zhou, Zhou; Huang, Yi; Han, Chengyun; Liu, Yan; Zhu, Qi; Ren, Chunmei

2016-09-01

Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

Case histories of bald eagles and other raptors killed by organophosphorus insecticides topically applied to livestock

USGS Publications Warehouse

Henny, C.J.; Kolbe, E.J.; Hill, E.F.; Blus, L.J.

1987-01-01

Since 1982 when secondary poisoning of a red-tailed hawk (Buteo jamaicensis) was documented following the recommended use of famphur applied topically to cattle, the Patuxent Wildlife Research Center has tested dead birds of prey for poisoning by famphur and other pour-on organophosphorus (OP) insecticides. Brain cholinesterase (ChE) activity was first determined, then if ChE was depressed greater than or equal to 50%, stomach and/or crop contents were evaluated for anti-ChE compounds. This report presents the circumstances surrounding the OP-caused deaths of eight bald eagles (Haliaeetus leucocephalus), two red-tailed hawks, and one great horned owl (Bubo virginianus) between March 1984 and March 1985. OP poisoning of raptors by pour-on insecticides in the United States is widespread, but its magnitude is unknown.

Case histories of bald eagles and other raptors killed by organophosphorus insecticides topically applied to livestock.

PubMed

Henny, C J; Kolbe, E J; Hill, E F; Blus, L J

1987-04-01

Since 1982 when secondary poisoning of a red-tailed hawk (Buteo jamaicensis) was documented following the recommended use of famphur applied topically to cattle, the Patuxent Wildlife Research Center has tested dead birds of prey for poisoning by famphur and other pour-on organophosphorus (OP) insecticides. Brain cholinesterase (ChE) activity was first determined, then if ChE was depressed greater than or equal to 50%, stomach and/or crop contents were evaluated for anti-ChE compounds. This report presents the circumstances surrounding the OP-caused deaths of eight bald eagles (Haliaeetus leucocephalus), two red-tailed hawks, and one great horned owl (Bubo virginianus) between March 1984 and March 1985. OP poisoning of raptors by pour-on insecticides in the United States is widespread, but its magnitude is unknown.

Putative bacterial volatile-mediated growth in soybean (Glycine max L. Merrill) and expression of induced proteins under salt stress.

PubMed

Vaishnav, A; Kumari, S; Jain, S; Varma, A; Choudhary, D K

2015-08-01

Plant root-associated rhizobacteria elicit plant immunity referred to as induced systemic tolerance (IST) against multiple abiotic stresses. Among multibacterial determinants involved in IST, the induction of IST and promotion of growth by putative bacterial volatile compounds (VOCs) is reported in the present study. To characterize plant proteins induced by putative bacterial VOCs, proteomic analysis was performed by MALDI-MS/MS after exposure of soybean seedlings to a new strain of plant growth promoting rhizobacteria (PGPR) Pseudomonas simiae strain AU. Furthermore, expression analysis by Western blotting confirmed that the vegetative storage protein (VSP), gamma-glutamyl hydrolase (GGH) and RuBisCo large chain proteins were significantly up-regulated by the exposure to AU strain and played a major role in IST. VSP has preponderant roles in N accumulation and mobilization, acid phosphatase activity and Na(+) homeostasis to sustain plant growth under stress condition. More interestingly, plant exposure to the bacterial strain significantly reduced Na(+) and enhanced K(+) and P content in root of soybean seedlings under salt stress. In addition, high accumulation of proline and chlorophyll content also provided evidence of protection against osmotic stress during the elicitation of IST by bacterial exposure. The present study reported for the first time that Ps. simiae produces a putative volatile blend that can enhance soybean seedling growth and elicit IST against 100 mmol l(-1) NaCl stress condition. The identification of such differentially expressed proteins provide new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to stresses. Further work should uncover more about the chemical side of VOC compounds and a detailed study about their molecular mechanism responsible for plant growth. © 2015 The Society for Applied Microbiology.

Occurrence of organophosphorus and carbamate pesticide residues in surface water samples from the Rangpur district of Bangladesh.

PubMed

Chowdhury, Alamgir Zaman; Jahan, Salina Akter; Islam, Mohammad Nazrul; Moniruzzaman, Mohammed; Alam, Mohammad Khorshed; Zaman, Mohammad A; Karim, Nurul; Gan, Siew Hua

2012-07-01

We report the presence of organophosphorus and carbamate residues in 24 surface water samples and five ground water samples from Pirgacha Thana, Rangpur district, Bangladesh using high-performance liquid chromatography. A number of samples of surface water from paddy fields were found to contain chlorpyriphos, carbofuran and carbaryl at concentrations ranging from 0-1.189, 0-3.395 and 0-0.163 μg/L, respectively. Surface water from the lakes had chlorpyriphos, carbofuran and carbaryl at concentrations ranging from 0.544-0.895, 0.949-1.671 and 0-0.195 μg/L, respectively. This result indicates that the general public living in the area of Rangpur is at high risk of pesticide exposure from contaminated waters in the environment.

Independent Prognostic Factors for Acute Organophosphorus Pesticide Poisoning.

PubMed

Tang, Weidong; Ruan, Feng; Chen, Qi; Chen, Suping; Shao, Xuebo; Gao, Jianbo; Zhang, Mao

2016-07-01

Acute organophosphorus pesticide poisoning (AOPP) is becoming a significant problem and a potential cause of human mortality because of the abuse of organophosphate compounds. This study aims to determine the independent prognostic factors of AOPP by using multivariate logistic regression analysis. The clinical data for 71 subjects with AOPP admitted to our hospital were retrospectively analyzed. This information included the Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, 6-h post-admission blood lactate levels, post-admission 6-h lactate clearance rates, admission blood cholinesterase levels, 6-h post-admission blood cholinesterase levels, cholinesterase activity, blood pH, and other factors. Univariate analysis and multivariate logistic regression analyses were conducted to identify all prognostic factors and independent prognostic factors, respectively. A receiver operating characteristic curve was plotted to analyze the testing power of independent prognostic factors. Twelve of 71 subjects died. Admission blood lactate levels, 6-h post-admission blood lactate levels, post-admission 6-h lactate clearance rates, blood pH, and APACHE II scores were identified as prognostic factors for AOPP according to the univariate analysis, whereas only 6-h post-admission blood lactate levels, post-admission 6-h lactate clearance rates, and blood pH were independent prognostic factors identified by multivariate logistic regression analysis. The receiver operating characteristic analysis suggested that post-admission 6-h lactate clearance rates were of moderate diagnostic value. High 6-h post-admission blood lactate levels, low blood pH, and low post-admission 6-h lactate clearance rates were independent prognostic factors identified by multivariate logistic regression analysis. Copyright © 2016 by Daedalus Enterprises.

Determination of selected pesticides in water samples adjacent to agricultural fields and removal of organophosphorus insecticide chlorpyrifos using soil bacterial isolates

NASA Astrophysics Data System (ADS)

Hossain, M. S.; Chowdhury, M. Alamgir Zaman; Pramanik, Md. Kamruzzaman; Rahman, M. A.; Fakhruddin, A. N. M.; Alam, M. Khorshed

2015-06-01

The use of pesticide for crops leads to serious environmental pollution, therefore, it is essential to monitor and develop approaches to remove pesticide from contaminated environment. In this study, water samples were collected to monitor pesticide residues, and degradation of chlorpyrifos was also performed using soil bacteria. Identification of pesticide residues and determination of their levels were performed by high-performance liquid chromatography with photodiode array detector. Among 12 samples, 10 samples were found contaminated with pesticides. Chlorpyrifos was detected in four tested samples and concentrations ranged from 3.27 to 9.31 μg/l whereas fenitrothion ranging from (Below Detection Limit, <0.1 μg/l) to 33.41 μg/l in the tested samples. Parathion was found in two tested samples at the concentration of 0.73 and 6.23 μg/l. None of the tested samples was found contaminated with Methoxychlor, DDT and Ethion. Three soil bacterial isolates, Pseudomonas peli BG1, Burkholderia caryophylli BG4 and Brevundimonas diminuta PD6 degraded chlorpyrifos completely in 8, 10 and 10 days, respectively, when 20 mg/l chlorpyrifos was supplied as sole source of carbon. Whereas, BG1, BG4 and PD6 took 14, 16 and 16 days, respectively, for complete removal of 50 mg/l chlorpyrifos. Chlorpyrifos degradation rates were found maximum by all three isolates at 2nd day of incubation for both tested concentrations. The results of the present study suggest the need for regular monitoring of pesticide residues in water, to protect the aquatic environment. Chlorpyrifos degrading bacterial isolates can be used to clean up environmental samples contaminated with the organophosphate pesticides.

Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ito, Tasuku; Saikawa, Kyo; Kim, Seonah

2014-04-25

Graphical abstract: - Highlights: • HypBA1 β-L-arabinofuranosidase belongs to glycoside hydrolase family 127. • Crystal structure of HypBA1 was determined. • HypBA1 consists of a catalytic barrel and two additional β-sandwich domains. • The active site contains a Zn{sup 2+} coordinated by glutamate and three cysteines. • A possible reaction mechanism involving cysteine as the nucleophile is proposed. - Abstract: Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-L-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-L-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system ofmore » hydroxyproline-linked β-L-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-L-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn{sup 2+} coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-L-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.« less

Expression of Nudix hydrolase genes in barley under UV irradiation

NASA Astrophysics Data System (ADS)

Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

Seed storage and cultivation should be necessary to self-supply foods when astronauts would stay and investigate during long-term space travel and habitation in the bases on the Moon and Mars. Thought the sunlight is the most importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development, UV presenting the sunlight can damage many aspects of plant processes at the physiological and DNA level. Especially UV-C, which is eliminated by the stratospheric ozone layer, is suspected to be extremely harmful and give a deadly injury to plants in space. However, the defense mechanism against UV-C irradiation damage in plant cells has not been clear. In this study, we investigated the expression of Nudix hydrolases, which defense plants from biotic / abiotic stress, in barley under UV irradiation. The genes encoding the amino acid sequences, which show homology to those of 28 kinds of Nudix hydrolases in Arabidopsis thaliana, were identified in the barley full-length cDNA library. BLAST analysis showed 14 kinds of barley genes (HvNUDX1-14), which encode the Nudix motif sequence. A phylogenetic tree showed that HvNUDX1, HvNUDX7, HvNUDX9 and HvNUDX11 belonged to the ADP-ribose pyrophosphohydrolase, ADP-sugar pyrophosphohydrolase, NAD(P)H pyrophosphohydrolase and FAD pyrophosphohydrolase subfamilies, respectively, HvNUDX3, HvNUDX6, and HvNUDX8 belonged to the Ap _{n}A pyrophosphohydrolase subfamilies, HvNUDX5 and HvNUDX14 belonged to the coenzyme A pyrophosphohydrolase subfamilies, HvNUDX12 and HvNUDX13 belonged to the Ap _{4}A pyrophosphohydrolase subfamilies. Induction of HvNUDX genes by UV-A (340nm), UV-B (312nm), and UV-C (260nm) were analyzed by quantitative RT-PCR. The results showed that HvNUDX4 was induced by UV-A and UV-B, HvNUDX6 was induced by UV-B and UV-C, and HvNUDX7 and HvNUDX14 were induced by UV-C, significantly. Our results suggest that the response of HvNUDXs to UV irradiation is different by UV

Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100

PubMed Central

Hashimoto, Masayuki; Fukui, Mitsuru; Hayano, Kouichi; Hayatsu, Masahito

2002-01-01

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon. PMID:11872471

Discovery of glycine sulfonamides as dual inhibitors of sn-1-diacylglycerol lipase α and α/β-hydrolase domain 6.

PubMed

Janssen, Freek J; Deng, Hui; Baggelaar, Marc P; Allarà, Marco; van der Wel, Tom; den Dulk, Hans; Ligresti, Alessia; van Esbroeck, Annelot C M; McGuire, Ross; Di Marzo, Vincenzo; Overkleeft, Herman S; van der Stelt, Mario

2014-08-14

sn-1-Diacylglycerol lipase α (DAGL-α) is the main enzyme responsible for the production of the endocannabinoid 2-arachidonoylglycerol in the central nervous system. Glycine sulfonamides have recently been identified by a high throughput screening campaign as a novel class of inhibitors for this enzyme. Here, we report on the first structure-activity relationship study of glycine sulfonamide inhibitors and their brain membrane proteome-wide selectivity on serine hydrolases with activity-based protein profiling (ABPP). We found that (i) DAGL-α tolerates a variety of biaryl substituents, (ii) the sulfonamide is required for inducing a specific orientation of the 2,2-dimethylchroman substituent, and (iii) a carboxylic acid is essential for its activity. ABPP revealed that the sulfonamide glycine inhibitors have at least three off-targets, including α/β-hydrolase domain 6 (ABHD6). Finally, we identified LEI-106 as a potent, dual DAGL-α/ABHD6 inhibitor, which makes this compound a potential lead for the discovery of new molecular therapies for diet-induced obesity and metabolic syndrome.

Trace Detection of Organophosphorus Chemical Warfare Agents in Wastewater and Plants by Luminescent UIO-67(Hf) and Evaluating the Bioaccumulation of Organophosphorus Chemical Warfare Agents.

PubMed

Lian, Xiao; Yan, Bing

2018-05-02

Organophosphorus chemical warfare agents (OPCWAs) are a group of organic pollutants characterized by high toxicity and chemical stability, and they are very difficult to be degraded. The trace quality of OPCWAs in water and food will cause great harm to the human body. Therefore, the detection of OPCWAs is a difficult challenge, which has become the research hotspot over the world. In this work, a Hf-based luminescent metal-organic framework (Eu@1) is prepared, and the reactivity of Hf 12 results in a methanephosphonic acid (MPA)-induced luminescence quenching and the charge transfer from MPA to Hf(IV) and generated exciplexes which are responsible for this quenching effect. The excellent performance of Eu@1 in the detection of MPA, with its finer selectivity, high sensitivity (LOD = 0.4 ppm), and large linear range (10 -7 to 10 -3 M), is encouraging for application in wastewater detection. Importantly, MPA is a pollutant that can be absorbed by plants and causes the bioaccumulation effect, and thus, the detection of MPA in real plant samples is a purposeful topic. Eu@1 also achieved satisfactory results in actual plant sample testing, and the bioaccumulation of MPA in onions, turnips, and cabbages is determined via our sensor. This fabricated detector provides a feasible path for the detection of ppm-level OPCWAs in a complex environment, which will help humans to avoid OPCWA-contaminated foods.

Zebrafish as a model for acetylcholinesterase-inhibiting organophosphorus agent exposure and oxime reactivation

PubMed Central

Koenig, Jeffrey A.; Dao, Thuy L.; Kan, Robert K.; Shih, Tsung-Ming

2016-01-01

The current research progression efforts for investigating novel treatments for exposure to organophosphorus (OP) compounds that inhibit acetylcholinesterase (AChE), including pesticides and chemical warfare nerve agents (CWNAs), rely solely on in vitro cell assays and in vivo rodent models. The zebrafish (Danio rerio) is a popular, well-established vertebrate model in biomedical research that offers high-throughput capabilities and genetic manipulation not readily available with rodents. A number of research studies have investigated the effects of subacute developmental exposure to OP pesticides in zebrafish, observing detrimental effects on gross morphology, neuronal development, and behavior. Few studies, however, have utilized this model to evaluate treatments, such as oxime reactivators, anticholinergics, or anticonvulsants, following acute exposure. Preliminary work has investigated the effects of CWNA exposure. The results clearly demonstrated relative toxicity and oxime efficacy similar to that reported for the rodent model. This review surveys the current literature utilizing zebrafish as a model for OP exposure and highlights its potential use as a high-throughput system for evaluating AChE reactivator antidotal treatments to acute pesticide and CWNA exposure. PMID:27123828

Cholinesterase reactivators and bioscavengers for pre- and post-exposure treatments of organophosphorus poisoning.

PubMed

Masson, Patrick; Nachon, Florian

2017-08-01

Organophosphorus agents (OPs) irreversibly inhibit acetylcholinesterase (AChE) causing a major cholinergic syndrome. The medical counter-measures of OP poisoning have not evolved for the last 30 years with carbamates for pretreatment, pyridinium oximes-based AChE reactivators, antimuscarinic drugs and neuroprotective benzodiazepines for post-exposure treatment. These drugs ensure protection of peripheral nervous system and mitigate acute effects of OP lethal doses. However, they have significant limitations. Pyridostigmine and oximes do not protect/reactivate central AChE. Oximes poorly reactivate AChE inhibited by phosphoramidates. In addition, current neuroprotectants do not protect the central nervous system shortly after the onset of seizures when brain damage becomes irreversible. New therapeutic approaches for pre- and post-exposure treatments involve detoxification of OP molecules before they reach their molecular targets by administrating catalytic bioscavengers, among them phosphotriesterases are the most promising. Novel generation of broad spectrum reactivators are designed for crossing the blood-brain barrier and reactivate central AChE. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Toxicology of organophosphorus compounds in view of an increasing terrorist threat.

PubMed

Worek, Franz; Wille, Timo; Koller, Marianne; Thiermann, Horst

2016-09-01

The implementation of the Chemical Weapon Convention (CWC), prohibiting the development, production, storage and use of chemical weapons by 192 nations and the ban of highly toxic OP pesticides, especially class I pesticides according to the WHO classification, by many countries constitutes a great success of the international community. However, the increased interest of terrorist groups in toxic chemicals and chemical warfare agents presents new challenges to our societies. Almost seven decades of research on organophosphorus compound (OP) toxicology was mainly focused on a small number of OP nerve agents despite the fact that a huge number of OP analogues, many of these agents having comparable toxicity to classical nerve agents, were synthesized and published. Only limited physicochemical, toxicological and medical information on nerve agent analogues is available in the open literature. This implies potential gaps of our capabilities to detect, to decontaminate and to treat patients if nerve agent analogues are disseminated and may result in inadequate effectiveness of newly developed countermeasures. In summary, our societies may face new, up to now disregarded, threats by toxic OP which calls for increased awareness and appropriate preparedness of military and civilian CBRN defense, a broader approach for new physical and medical countermeasures and an integrated system of effective detection, decontamination, physical protection and treatment.

Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior

PubMed Central

Masson, Patrick; Lockridge, Oksana

2009-01-01

Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include a) treatment with an OP scavenger, b) reaction of nonaged enzyme with oximes, c) reactivation of aged enzyme, d) slowing down aging with peripheral site ligands, and e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methyl indoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine. PMID:20004171

Modeling receptor kinetics in the analysis of survival data for organophosphorus pesticides.

PubMed

Jager, Tjalling; Kooijman, Sebastiaan A L M

2005-11-01

Acute ecotoxicological tests usually focus on survival at a standardized exposure time. However, LC50's decrease in time in a manner that depends both on the chemical and on the organism. DEBtox is an existing approach to analyze toxicity data in time, based on hazard modeling (the internal concentration increases the probability to die). However, certain chemicals elicit their response through (irreversible) interaction with a specific receptor, such as inhibition of acetylcholinesterase (AChE). Effects therefore do not solely depend on the actual internal concentration, but also on its (recent) past. In this paper, the DEBtox method is extended with a simple mechanistic model to deal with receptor interactions. We analyzed data from the literature for organophosphorus pesticides in guppies, fathead minnows, and springtails. Overall, the observed survival patterns do not clearly differ from those of chemicals with a less-specific mode of action. However, using the receptor model, resulting parameter estimates are easier to interpret in terms of underlying mechanisms and reveal similarities between the various pesticides. We observed thatthe no-effect concentration estimated from the receptor model is basically identical to the value from standard DEBtox, illustrating the robustness of this summary statistic.

An Enhanced Butyrylcholinesterase Method to Measure Organophosphorus Nerve Agent Exposure in Humans

PubMed Central

Pantazides, Brooke G.; Watson, Caroline M.; Carter, Melissa D.; Crow, Brian S.; Perez, Jonas W.; Blake, Thomas A.; Thomas, Jerry D.; Johnson, Rudolph C.

2016-01-01

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be used to confirm exposure in humans. A highly accurate method to detect G-series and V-series OPNA adducts to BChE in 75 μL of filtered blood, serum, or plasma has been developed using immunomagnetic separation (IMS) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The reported IMS method captures > 88% of the BChE in a specimen and corrects for matrix effects on peptide calibrators. The optimized method has been used to quantify baseline BChE levels (unadducted and OPNA-adducted) in a matched set of serum, plasma and whole blood (later processed in-house for plasma content) from 192 unexposed individuals to determine the interchangeability of the tested matrices. The results of these measurements demonstrate the ability to accurately measure BChE regardless of the format of the blood specimen received. Criteria for accepting or denying specimens were established through a series of sample stability and processing experiments. The results of these efforts are an optimized and rugged method that is transferrable to other laboratories and an increased understanding of the BChE biomarker in matrix. PMID:24604326

Degradation of Organophosphorus and Pyrethroid Insecticides in Beverages: Implications for Risk Assessment.

PubMed

Radford, Samantha A; Panuwet, Parinya; Hunter, Ronald E; Barr, Dana Boyd; Ryan, P Barry

2018-02-02

Since urinary insecticide metabolites are commonly used as biomarkers of exposure, it is important that we quantify whether insecticides degrade in food and beverages in order to better perform risk assessment. This study was designed to quantify degradation of organophosphorus and pyrethroid insecticides in beverages. Purified water, white grape juice, orange juice, and red wine were fortified with 500 ng/mL diazinon, malathion, chlorpyrifos, permethrin, cyfluthrin, cypermethrin, and deltamethrin, and aliquots were extracted several times over a 15-day storage period at 2.5 °C. Overall, statistically significant loss of at least one insecticide was observed in each matrix, and at least five out of seven insecticides demonstrated a statistically significant loss in all matrices except orange juice. An investigation of an alternative mechanism of insecticide loss-adsorption onto the glass surface of the storage jars-was carried out, which indicated that this mechanism of loss is insignificant. Results of this work suggest that insecticides degrade in these beverages, and this degradation may lead to pre-existing insecticide degradates in the beverages, suggesting that caution should be exercised when using urinary insecticide metabolites to assess exposure and risk.

Degradation of Organophosphorus and Pyrethroid Insecticides in Beverages: Implications for Risk Assessment

PubMed Central

Panuwet, Parinya; Hunter, Ronald E.; Barr, Dana Boyd; Ryan, P. Barry

2018-01-01

Since urinary insecticide metabolites are commonly used as biomarkers of exposure, it is important that we quantify whether insecticides degrade in food and beverages in order to better perform risk assessment. This study was designed to quantify degradation of organophosphorus and pyrethroid insecticides in beverages. Purified water, white grape juice, orange juice, and red wine were fortified with 500 ng/mL diazinon, malathion, chlorpyrifos, permethrin, cyfluthrin, cypermethrin, and deltamethrin, and aliquots were extracted several times over a 15-day storage period at 2.5 °C. Overall, statistically significant loss of at least one insecticide was observed in each matrix, and at least five out of seven insecticides demonstrated a statistically significant loss in all matrices except orange juice. An investigation of an alternative mechanism of insecticide loss—adsorption onto the glass surface of the storage jars—was carried out, which indicated that this mechanism of loss is insignificant. Results of this work suggest that insecticides degrade in these beverages, and this degradation may lead to pre-existing insecticide degradates in the beverages, suggesting that caution should be exercised when using urinary insecticide metabolites to assess exposure and risk. PMID:29393904

Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, Johnnie A.; Pattathil, Sivakumar; Bergeman, Lai F.

Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions. Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss ofmore » specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides. The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.« less

A new high-performance thin-layer chromatographic method for determining bile salt hydrolase activity.

PubMed

Rohawi, Nur Syakila; Ramasamy, Kalavathy; Agatonovic-Kustrin, Snezana; Lim, Siong Meng

2018-06-05

A quantitative assay using high-performance thin-layer chromatography (HPTLC) was developed to investigate bile salt hydrolase (BSH) activity in Pediococcus pentosaceus LAB6 and Lactobacillus plantarum LAB12 probiotic bacteria isolated from Malaysian fermented food. Lactic acid bacteria (LAB) were cultured in de Man Rogosa and Sharpe (MRS) broth containing 1 mmol/L of sodium-based glyco- and tauro-conjugated bile salts for 24 h. The cultures were centrifuged and the resultant cell free supernatant was subjected to chromatographic separation on a HPTLC plate. Conjugated bile salts were quantified by densitometric scans at 550 nm and results were compared to digital image analysis of chromatographic plates after derivatisation with anisaldehyde/sulfuric acid. Standard curves for bile salts determination with both methods show good linearity with high coefficient of determination (R 2 ) between 0.97 and 0.99. Method validation indicates good sensitivity with low relative standard deviation (RSD) (<10%), low limits of detection (LOD) of 0.4 versus 0.2 μg and limit of quantification (LOQ) of 1.4 versus 0.7 μg, for densitometric vs digital image analysis method, respectively. The bile salt hydrolase activity was found to be higher against glyco- than tauro-conjugated bile salts (LAB6; 100% vs >38%: LAB12; 100% vs >75%). The present findings strongly show that quantitative analysis via digitally-enhanced HPTLC offers a rapid quantitative analysis for deconjugation of bile salts by probiotics. Copyright © 2018. Published by Elsevier B.V.

Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling

DOE PAGES

Walker, Johnnie A.; Pattathil, Sivakumar; Bergeman, Lai F.; ...

2017-02-02

Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions. Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss ofmore » specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides. The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.« less

Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol using a novel bacterium Ochrobactrum sp. JAS2: A proposal of its metabolic pathway.

PubMed

Abraham, Jayanthi; Silambarasan, Sivagnanam

2016-01-01

Biodegradation of chlorpyrifos and its major metabolite 3,5,6-trichloro-2-pyridinol (TCP) were studied with a novel bacterial strain JAS2 isolated from paddy rhizosphere soil. The molecular characterization based on 16S rRNA gene sequence homology confirmed its identity as Ochrobactrum sp. JAS2. The JAS2 strain degraded 300mgl(-1) of chlorpyrifos within 12h of incubation in the aqueous medium and it produced the TCP metabolite. However, after 72h of incubation TCP was also completely degraded by the JAS2 strain. A tentative degradation pathway of chlorpyrifos by Ochrobactrum sp. JAS2 has been proposed on basis of GC-MS analysis. The complete degradation of chlorpyrifos occurred within 24h in the soil spiked with and without addition of nutrients inoculated with Ochrobactrum sp. JAS2. TCP was obtained in both the studies which was degraded completely by 96h in the soil spiked with nutrients and whereas 120h in absence of nutrients in the soil. The mpd gene which is responsible for organophosphorus hydrolase production was identified. The isolates Ochrobactrum sp. JAS2 also exhibited a time dependent increase in the amount of tricalcium phosphate solubilization in Pikovskaya's medium. Further screening of the strain JAS2 for auxiliary plant growth promoting activities revealed its remarkable capability of producing the indole acetic acid (IAA), hydrogen cyanide (HCN) and ammonia. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

PubMed

Duan, Cheng-Jie; Huang, Ming-Yue; Pang, Hao; Zhao, Jing; Wu, Chao-Xing; Feng, Jia-Xun

2017-07-01

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat /K M ) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

Acute effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate, on cardiovascular parameters in anaesthetized, artificially ventilated rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Yoshimasa; Itoh, Takeo, E-mail: titoh@med.nagoya-cu.ac.jp; Shiraishi, Hiroaki

The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8 mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolaminemore » (non-selective α-adrenergic receptor antagonist) plus propranolol (non-selective β-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1 mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP. - Highlights: • A sarin-like agent BIMP markedly increased blood pressure in anaesthetized rats. • Muscarinic receptor blockade enhanced the BIMP-induced increase in blood pressure. • Ganglionic nicotinic receptor blockade attenuated the BIMP-induced response. • Blockade of α- as well as β-receptors attenuated the BIMP-induced response.« less

Metabolism of Phloridzin by Erwinia herbicola: Nature of the Degradation Products, and the Purification and Properties of Phloretin Hydrolase

PubMed Central

Chatterjee, A. K.; Gibbins, L. N.

1969-01-01

Erwinia herbicola Y46 degrades phloridzin to yield phloretin, phloroglucinol, and phloretic acid, when grown on defined medium containing phloridzin as the sole source of carbon. The identities of the intermediates isolated from culture filtrates were established by co-chromatography and by ultraviolet absorption spectra. Only 3 of 11 strains of this species, and none of the 12 species of bacterial phytopathogens tested could effect this breakdown. Some of the latter organisms possessed β-glucosidase activity which liberated d-glucose from phloridzin. The enzyme phloretin hydrolase was purified from cells of E. herbicola Y46 grown on Yeast Beef Broth, by treatment of crude extracts with protamine sulfate, ammonium sulfate precipitation, elution from calcium phosphate gel, elution from diethylaminoethyl-cellulose, and concentration by ultrafiltration. The final preparation was free of β-glucosidase, had a specific activity of 213 units per mg of protein, and represented a 142-fold purification over the crude extract. The enzyme had a pH optimum of 6.7 to 6.8, and produced only phloroglucinol and phloretic acid as products of phloretin breakdown, there being an equimolar relationship between the cleavage of phloretin and the formation of the products. The Michaelis constant (Km) for the enzyme with phloretin as substrate was 3.8 × 10−5m, and the enzyme was sensitive to Hg2+ and Cu2+ ions. Phloroglucinol, phloretic acid, p-chloromercuribenzoate and iodoacetamide were without effect on the activity. The enzyme did not react with phloridzin, naringin, or naringenin. The physiological significance of the results is discussed. PMID:5354935

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases.

PubMed Central

Srivastava, A; Jones, E W

1998-01-01

The PEP12 homolog Pth1p (Pep twelve homolog 1) is predicted to be similar in size to Pep12p, the endosomal syntaxin homolog that mediates docking of Golgi-derived transport vesicles and, like other members of the syntaxin family, is predicted to be a cytoplasmically oriented, integral membrane protein with a C-terminal transmembrane domain. Kinetic analyses indicate that deltapth1/vam3 mutants fail to process the soluble vacuolar hydrolase precursors and that PrA, PrB and most of CpY accumulate within the cell in their Golgi-modified P2 precursor forms. This is in contrast to a pep12 mutant in which P2CpY is secreted from the cell. Furthermore, pep12 is epistatic to pth1/vam3 with respect to the CpY secretion phenotype. Alkaline phosphatase, a vacuolar membrane hydrolase, accumulates in its precursor form in the deltapth1/vam3 mutant. Maturation of pro-aminopeptidase I, a hydrolase precursor delivered directly to the vacuole from the cytoplasm, is also blocked in the deltapth1/vam3 mutant. Subcellular fractionation localizes Pth1/Vam3p to vacuolar membranes. Based on these data, we propose that Pth1/Vam3p is the vacuolar syntaxin/t-SNARE homolog that participates in docking of transport vesicles at the vacuolar membrane and that the function of Pth1/Vam3p impinges on at least three routes of protein delivery to the yeast vacuole. PMID:9475723

Unveiling the functional diversity of the Alpha-Beta hydrolase fold in plants

PubMed Central

Mindrebo, Jeffrey T.; Nartey, Charisse M.; Seto, Yoshiya; Burkart, Michael D.; Noel, Joseph P.

2017-01-01

The alpha/beta hydrolase (ABH) superfamily is a widespread and functionally malleable protein fold recognized for its diverse biochemical activities across all three domains of life. ABH enzymes possess unexpected catalytic activity in the green plant lineage through selective alterations in active site architecture and chemistry. Furthermore, the ABH fold serves as the core structure for phytohormone and ligand receptors in the gibberellin, strigolactone, and karrikin signaling pathways in plants. Despite recent discoveries, the ABH family is sparsely characterized in plants, a sessile kingdom known to evolve complex and specialized chemical adaptations as survival responses to widely varying biotic and abiotic ecologies. This review calls attention to the ABH superfamily in the plant kingdom to highlight the functional adaptability of the ABH fold. PMID:27662376

Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

PubMed

Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

2012-01-15

Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Selective Inhibition of Plant Serine Hydrolases by Agrochemicals Revealed by Competitive ABPP

PubMed Central

Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F.; Kaiser, Markus; van der Hoorn, Renier A. L.

2013-01-01

Organophosphate and –phosphonates and their thiol derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant and their consumers are poorly investigated. Here, we use competitive Activity-based Protein Profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. PMID:21764588

In situ ionic liquid dispersive liquid-liquid microextraction coupled to gas chromatography-mass spectrometry for the determination of organophosphorus pesticides.

PubMed

Cacho, J I; Campillo, N; Viñas, P; Hernández-Córdoba, M

2018-07-20

Nine organophosphorus pesticides (OPPs) were determined in environmental waters from different origins using in situ ionic liquid dispersive liquid microextraction (IL-DLLME). This preconcentration technique was coupled to gas chromatography-mass spectrometry (GC-MS) using microvial insert thermal desorption, an approach that uses a thermal desorption injector as sample introduction system. The parameters affecting both the microextraction and sample injection steps were optimized. The proposed method showed good precision, with RSD values ranging from 4.1 to 9.7%, accuracy with recoveries in the 85-118% range, and sensitivity with DLs ranging from 5 to 16 ng L -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

Photodegradation of organophosphorus pesticides in honey medium.

PubMed

Yuan, Zhimin; Yao, Jun; Liu, Haijun; Han, Jun; Trebše, Polonca

2014-10-01

Honey can be polluted due to environmental pollution and misuse of beekeeping practices. In the present study, photodegradation experiments of organophosphorus pesticides (coumaphos, methyl parathion and fenitrothion) in honey medium were conducted using Atlas Suntest simulator CPS+ as a sunlight producer. Photodegradation experiments were conducted under three different intensities as 250W/m(2), 500W/m(2) and 750W/m(2) to evaluate the impact of sunlight intensity on removal of OPs in honey medium. Significant decreases of three OPs' concentrations were observed. Coumaphos showed the highest degradability, reaching a degradation percentage of 90 percent within 15min. After 1h irradiation, residual percentages of coumaphos were 6.62 percent for 250W/m(2), 3.48 percent for 500W/m(2) and 2.98 percent for 750W/m(2), respectively. Methyl parathion and fenitrothion also could be removed through photodegradation efficiently. After 1h irradiation, the residual percentages of methyl parathion and fenitrothion under 750W/m(2) sunlight irradiation were 26.89 percent and 16.70 percent, respectively. Intensity of sunlight showed a positive impact on removal of OPs in honey medium. The higher intensity, the lower residual percentage. Photodegradation of three OPs fitted well with pseudo-first order kinetics. Half-lives calculated from pseudo-first order kinetics were 17.61min (250W/m(2)), 16.67min (500W/m(2)) and 17.58min (750W/m(2)) for coumaphos, 57.62min (250W/m(2)), 34.13min (500W/m(2)) and 31.69min (750W/m(2)) for methyl parathion and 144.70min (250W/m(2)), 95.47min (500W/m(2)) and 22.57min (750W/m(2)) for fenitrothion, respectively. Most of the three OPs could dissipate in a short time under sunlight irradiation. Photodegradation could be accepted as an appropriate method for the removal of OPs in honey medium. Copyright © 2014 Elsevier Inc. All rights reserved.