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1.
PLoS One ; 17(4): e0267509, 2022.
Article in English | MEDLINE | ID: mdl-35452491

ABSTRACT

ß-Mannans are a heterogeneous group of polysaccharides with a common main chain of ß-1,4-linked mannopyranoside residues. The cleavage of ß-mannan chains is catalyzed by glycoside hydrolases called ß-mannanases. In the CAZy database, ß-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.


Subject(s)
Firmicutes , Glycoside Hydrolases , Mannans , Firmicutes/enzymology , Firmicutes/genetics , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Mannans/chemistry , Mannose , Substrate Specificity , beta-Mannosidase/metabolism
2.
Carbohydr Res ; 515: 108544, 2022 May.
Article in English | MEDLINE | ID: mdl-35367699

ABSTRACT

Noctoc commune is a cyanobacterium living in various and extreme environments. Its ability to survive in desert, on ice or high altitude is explained by its exceptional metabolism and its capacity to resist to desiccation. N. commune cells are embedded in a gelatinous matrix made of polysaccharides which fixes water and participates in maintaining the cells in hydrated conditions. The structure of the polysaccharide of N. commune harvested in Saint Martin d'Uriage (France) and the oligosaccharides obtained after its enzymatic degradation were determined. The repeating unit of the main chain is a tetra-saccharide: [→4)-ß-D-Glcp-(1 â†’ 4)-ß-D-Xylp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-α-D-Galp-(1→], branched at position 6 of a glucose residue by a ß-linked pyruvated glucuronic acid residue. About 30% of the Xylp residues were branched with a Xylf residue. Comparison of this structure with the polysaccharides secreted by other Nostoc species and strains suggest a strong selection pressure on the structure in agreement with its important biological role.


Subject(s)
Nostoc commune , Carbohydrates , Oligosaccharides/chemistry , Polysaccharides/chemistry , Water
3.
Proc Natl Acad Sci U S A ; 116(13): 6063-6068, 2019 03 26.
Article in English | MEDLINE | ID: mdl-30850540

ABSTRACT

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.


Subject(s)
Carbohydrate Metabolism , Enzymes/genetics , Sequence Analysis, Protein , Amino Acid Sequence , Animals , Carbohydrate Metabolism/genetics , Enzymes/metabolism , Genomics/methods , Humans , Polysaccharides/metabolism , Sequence Analysis, DNA , Structure-Activity Relationship
4.
Sci Rep ; 8(1): 8075, 2018 05 23.
Article in English | MEDLINE | ID: mdl-29795267

ABSTRACT

In bacteria from the phylum Bacteroidetes, the genes coding for enzymes involved in polysaccharide degradation are often colocalized and coregulated in so-called "polysaccharide utilization loci" (PULs). PULs dedicated to the degradation of marine polysaccharides (e.g. laminaran, ulvan, alginate and porphyran) have been characterized in marine bacteria. Interestingly, the gut microbiome of Japanese individuals acquired, by lateral transfer from marine bacteria, the genes involved in the breakdown of porphyran, the cell wall polysaccharide of the red seaweed used in maki. Sequence similarity analyses predict that the human gut microbiome also encodes enzymes for the degradation of alginate, the main cell wall polysaccharide of brown algae. We undertook the functional characterization of diverse polysaccharide lyases from family PL17, frequently found in marine bacteria as well as those of human gut bacteria. We demonstrate here that this family is polyspecific. Our phylogenetic analysis of family PL17 reveals that all alginate lyases, which have all the same specificity and mode of action, cluster together in a very distinct subfamily. The alginate lyases found in human gut bacteria group together in a single clade which is rooted deeply in the PL17 tree. These enzymes were found in PULs containing PL6 enzymes, which also clustered together in the phylogenetic tree of PL6. Together, biochemical and bioinformatics analyses suggest that acquisition of this system appears ancient and, because only traces of two successful transfers were detected upon inspection of PL6 and PL17 families, the pace of acquisition of marine polysaccharide degradation system is probably very slow.


Subject(s)
Alginates/metabolism , Bacteria/metabolism , Gastrointestinal Microbiome , Polysaccharide-Lyases/metabolism , Bacteria/genetics , Gene Expression Regulation, Bacterial , Humans , Multigene Family , Phylogeny , Polysaccharide-Lyases/genetics , Substrate Specificity
5.
Chem Biol Interact ; 267: 11-16, 2017 Apr 01.
Article in English | MEDLINE | ID: mdl-26972668

ABSTRACT

Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 µmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice.


Subject(s)
Blood Chemical Analysis/methods , Blood-Brain Barrier/drug effects , Chemical Warfare Agents/toxicity , Cholinesterase Reactivators/blood , Organothiophosphorus Compounds/toxicity , Oximes/pharmacology , Pralidoxime Compounds/pharmacology , Pyridinium Compounds/pharmacology , Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Animals , Blood-Brain Barrier/metabolism , Erythrocytes/cytology , Erythrocytes/enzymology , Half-Life , Humans , Injections, Intraperitoneal , Male , Mice , Oximes/metabolism , Pralidoxime Compounds/metabolism , Protective Agents/metabolism , Protective Agents/pharmacology , Pyridinium Compounds/metabolism
6.
Eur J Med Chem ; 78: 455-67, 2014 May 06.
Article in English | MEDLINE | ID: mdl-24704618

ABSTRACT

A series of new uncharged functional acetylcholinesterase (AChE) reactivators including heterodimers of tetrahydroacridine with 3-hydroxy-2-pyridine aldoximes and amidoximes has been synthesized. These novel molecules display in vitro reactivation potencies towards VX-, tabun- and paraoxon-inhibited human AChE that are superior to those of the mono- and bis-pyridinium aldoximes currently used against nerve agent and pesticide poisoning. Furthermore, these uncharged compounds exhibit a broader reactivity spectrum compared to currently approved remediation drugs.


Subject(s)
Acetylcholinesterase/metabolism , Chemical Warfare Agents/pharmacology , Cholinesterase Inhibitors/pharmacology , Drug Design , Amides/chemistry , Amides/pharmacology , Chemical Warfare Agents/chemical synthesis , Chemical Warfare Agents/chemistry , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Oximes/chemistry , Oximes/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Structure-Activity Relationship , Tacrine/chemistry , Tacrine/pharmacology
7.
Chem Commun (Camb) ; 50(30): 3947-50, 2014 Apr 18.
Article in English | MEDLINE | ID: mdl-24599312

ABSTRACT

Two promising uncharged reactivators for inhibited human BChE and AChE have been described. These compounds show an ability to reactivate VX-inhibited BChE largely superior to those of known pyridinium aldoximes. Moreover, these oximes also exhibit a good ability to reactivate VX-, tabun- and paraoxon-inhibited human AChE.


Subject(s)
Acetylcholinesterase/metabolism , Butyrylcholinesterase/metabolism , Carbolines/pharmacology , Cholinesterase Inhibitors/pharmacology , Oximes/pharmacology , Carbolines/chemical synthesis , Carbolines/chemistry , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Humans , Molecular Structure , Oximes/chemical synthesis , Oximes/chemistry , Phosphorylation/drug effects , Structure-Activity Relationship
8.
Chem Biol Interact ; 203(1): 19-23, 2013 Mar 25.
Article in English | MEDLINE | ID: mdl-22922115

ABSTRACT

Organophosphorus nerve agents irreversibly inhibit cholinesterases. Phosphylation of the catalytic serine can be reversed by the mean of powerful nucleophiles like oximes. But the phosphyl adduct can undergo a rapid spontaneous reaction leading to an aged enzyme, i.e., a conjugated enzyme that is no longer reactivable by oximes. One strategy to regain reactivability is to alkylate the phosphylic adduct. Specific alkylating molecules were synthesized and the crystal structures of the complexes they form with soman-aged human butyrylcholinesterase were solved. Although the compounds bind in the active site gorge of the aged enzyme, the orientation of the alkylating function appears to be unsuitable for efficient alkylation of the phosphylic adduct. However, these crystal structures provide key information to design efficient alkylators of aged-butyrylcholinesterase and specific reactivators of butyrylcholinesterase.


Subject(s)
Butyrylcholinesterase/chemistry , Butyrylcholinesterase/metabolism , Alkylation , Catalytic Domain , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/toxicity , Cholinesterase Reactivators/pharmacology , Crystallography, X-Ray , Humans , Kinetics , Ligands , Models, Molecular , Phosphorylation , Pralidoxime Compounds/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine/chemistry , Soman/toxicity
9.
Chem Biol Interact ; 203(1): 81-4, 2013 Mar 25.
Article in English | MEDLINE | ID: mdl-23111374

ABSTRACT

Organophosphorus nerve agents (OPNAs) are highly toxic compounds that represent a threat to both military and civilian populations. They cause an irreversible inhibition of acetylcholinesterase (AChE), by the formation of a covalent P-O bond with the catalytic serine. Among the present treatment of nerve agents poisoning, pyridinium and bis-pyridinium aldoximes are used to reactivate this inhibited enzyme but these compounds do not readily cross the blood brain barrier (BBB) due to their permanent cationic charge and thus cannot efficiently reactivate cholinesterases in the central nervous system (CNS). In this study, a series of seven new uncharged oximes reactivators have been synthesized and their in vitro ability to reactivate VX and tabun-inhibited human acetylcholinesterase (hAChE) has been evaluated. The dissociation constant K(D) of inhibited enzyme-oxime complex, the reactivity rate constant kr and the second order reactivation rate constant k(r2) have been determined and have been compared to reference oximes HI-6, Obidoxime and 2-Pralidoxime (2-PAM). Regarding the reactivation of VX-inhibited hAChE, all compounds show a better reactivation potency than those of 2-PAM, nevertheless they are less efficient than obidoxime and HI-6. Moreover, one of seven described compounds presents an ability to reactivate tabun-inhibited hAChE equivalent to those of 2-PAM.


Subject(s)
Acetylcholinesterase/metabolism , Chemical Warfare Agents/toxicity , Cholinesterase Inhibitors/toxicity , Cholinesterase Reactivators/chemical synthesis , Cholinesterase Reactivators/pharmacology , Organophosphorus Compounds/toxicity , Cholinesterase Reactivators/chemistry , Drug Evaluation, Preclinical , Electrochemistry , GPI-Linked Proteins/metabolism , Humans , Molecular Structure , Obidoxime Chloride/pharmacology , Oximes/chemical synthesis , Oximes/chemistry , Oximes/pharmacology , Pralidoxime Compounds/pharmacology , Pyridinium Compounds/pharmacology , Recombinant Proteins/metabolism
10.
J Med Chem ; 55(23): 10791-5, 2012 Dec 13.
Article in English | MEDLINE | ID: mdl-23148598

ABSTRACT

Pyridinium and bis-pyridinium aldoximes are used as antidotes to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus nerve agents. Herein, we described a series of nine nonquaternary phenyltetrahydroisoquinoline-pyridinaldoxime conjugates more efficient than or as efficient as pyridinium oximes to reactivate VX-, tabun- and ethyl paraoxon-inhibited human AChE. This study explores the structure-activity relationships of this new family of reactivators and shows that 1b-d are uncharged hAChE reactivators with a broad spectrum.


Subject(s)
Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Enzyme Reactivators/pharmacology , Isoquinolines/pharmacology , Oximes/pharmacology , Acetylcholinesterase/drug effects , Humans , Magnetic Resonance Spectroscopy , Phosphorylation
11.
Toxicol Lett ; 206(1): 14-23, 2011 Sep 25.
Article in English | MEDLINE | ID: mdl-21683774

ABSTRACT

Bioscavengers are molecules able to neutralize neurotoxic organophosphorus compounds (OP) before they can reach their biological target. Human butyrylcholinesterase (hBChE) is a natural bioscavenger each molecule of enzyme neutralizing one molecule of OP. The amount of natural enzyme is insufficient to achieve good protection. Thus, different strategies have been envisioned. The most straightforward consists in injecting a large dose of highly purified natural hBChE to increase the amount of bioscavenger in the bloodstream. This proved to be successful for protection against lethal doses of soman and VX but remains expensive. An improved strategy is to regenerate prophylactic cholinesterases (ChE) by administration of reactivators after exposure. But broad-spectrum efficient reactivators are still lacking, especially for inhibited hBChE. Cholinesterase mutants capable of reactivating spontaneously are another option. The G117H hBChE mutant has been a prototype. We present here the Y124H/Y72D mutant of human acetylcholinesterase; its spontaneous reactivation rate after V-agent inhibition is increased up to 110 fold. Catalytic bioscavengers, enzymes capable of hydrolyzing OP, present the best alternative. Mesophilic bacterial phosphotriesterase (PTE) is a candidate with good catalytic efficiency. Its enantioselectivity has been enhanced against the most potent OP isomers by rational design. We show that PEGylation of this enzyme improves its mean residence time in the rat blood stream 24-fold and its bioavailability 120-fold. Immunogenic issues remain to be solved. Human paraoxonase 1 (hPON1) is another promising candidate. However, its main drawback is that its phosphotriesterase activity is highly dependent on its environment. Recent progress has been made using a mammalian chimera of PON1, but we provide here additional data showing that this chimera is biochemically different from hPON1. Besides, the chimera is expected to suffer from immunogenic issues. Thus, we stress that interest for hPON1 must not fade away, and in particular, the 3D structure of the hPON1 eventually in complex with OP has to be solved.


Subject(s)
Acetylcholinesterase/pharmacology , Aryldialkylphosphatase/pharmacology , Biocatalysis , Cholinesterase Reactivators/pharmacology , Organophosphorus Compounds/chemistry , Phosphoric Triester Hydrolases/pharmacology , Acetylcholinesterase/genetics , Acetylcholinesterase/metabolism , Animals , Aryldialkylphosphatase/blood , Aryldialkylphosphatase/metabolism , CHO Cells , Chemical Warfare Agents/chemistry , Chemical Warfare Agents/toxicity , Cholinesterase Reactivators/blood , Cholinesterase Reactivators/metabolism , Cloning, Molecular , Cricetinae , Cricetulus , Drug Stability , Female , Hydrolysis , Mutation , Organophosphorus Compounds/toxicity , Phosphoric Triester Hydrolases/metabolism , Rats , Rats, Wistar , Substrate Specificity , Transfection
12.
Chem Biol Interact ; 187(1-3): 380-3, 2010 Sep 06.
Article in English | MEDLINE | ID: mdl-20230809

ABSTRACT

Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.


Subject(s)
Biocatalysis , Organophosphate Poisoning , Organophosphates/metabolism , Phosphoric Triester Hydrolases/chemistry , Phosphoric Triester Hydrolases/metabolism , Polyethylene Glycols/chemistry , Aldehydes/chemistry , Antidotes/chemistry , Antidotes/isolation & purification , Antidotes/metabolism , Antidotes/pharmacology , Caulobacteraceae/enzymology , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzyme Stability , Hydrogen-Ion Concentration , Phosphoric Triester Hydrolases/isolation & purification , Phosphoric Triester Hydrolases/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature
13.
J Enzyme Inhib Med Chem ; 24(4): 1045-55, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19548794

ABSTRACT

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co(2+)-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn(2+) whereas Co-PTE has Zn(2+) and Co(2+) in equimolar amount, with Co(2+) most likely in the reported labile beta-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl(2) induces a non-competitive partial inhibition of Zn(2+)- and Co(2+)-PTE at pH 8.5 (apparent Ki=155 microM and 52 microM, respectively). Inhibition results from interactions with colloidal Zn(OH)(2) formed in alkaline buffer that alters the catalytic machinery. NiCl(2) caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co(2+)-PTE, Ki for Zn(OH)(2) inhibition, k(cat) (up to 60 fold) for paraoxon hydrolysis, but not K(M). Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.


Subject(s)
Phosphoric Triester Hydrolases/chemistry , Pseudomonas/enzymology , Recombinant Proteins , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Kinetics , Mass Spectrometry , Models, Molecular , Molecular Structure , Phosphoric Triester Hydrolases/genetics , Phosphoric Triester Hydrolases/metabolism , Pseudomonas/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, X-Ray Emission
14.
Biochem J ; 421(1): 97-106, 2009 Jun 12.
Article in English | MEDLINE | ID: mdl-19368529

ABSTRACT

hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4 [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HLö 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.


Subject(s)
Butyrylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Cholinesterase Reactivators/pharmacology , Organophosphates/pharmacology , Oximes/pharmacology , Catalytic Domain , Cholinesterase Inhibitors/chemistry , Cholinesterase Reactivators/chemistry , Humans , Kinetics , Models, Molecular , Molecular Structure , Oximes/chemistry , Protein Conformation , Structure-Activity Relationship , Time Factors
15.
J Am Chem Soc ; 130(47): 16011-20, 2008 Nov 26.
Article in English | MEDLINE | ID: mdl-18975951

ABSTRACT

Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.


Subject(s)
Cholinesterases/chemistry , Cholinesterases/metabolism , Organophosphates/chemistry , Alkylation , Catalytic Domain , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Crystallography, X-Ray , Humans , Mass Spectrometry , Models, Molecular , Molecular Structure , Phosphorylation
16.
Biochim Biophys Acta ; 1764(9): 1470-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16962835

ABSTRACT

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.


Subject(s)
Acetylcholinesterase/genetics , Acetylcholinesterase/metabolism , Organophosphorus Compounds/metabolism , Acetylthiocholine/metabolism , Acetylthiocholine/pharmacology , Animals , Bungarus , Chlorpyrifos/analogs & derivatives , Chlorpyrifos/pharmacology , Disulfoton/pharmacology , Echothiophate Iodide/metabolism , Echothiophate Iodide/pharmacology , Isoflurophate/metabolism , Isoflurophate/pharmacology , Mutagenesis, Site-Directed , Mutation , Paraoxon/metabolism , Paraoxon/pharmacology
17.
J Exp Med ; 196(9): 1163-73, 2002 Nov 04.
Article in English | MEDLINE | ID: mdl-12417627

ABSTRACT

Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR alpha repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3' side than 5' end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.


Subject(s)
Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Receptors, Antigen, T-Cell, alpha-beta/genetics , Thymus Gland/cytology , Animals , Cell Differentiation , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor/genetics , Mice , Mice, Inbred BALB C
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