Carr PD

References (14)

Title : Structure of an Insecticide Sequestering Carboxylesterase from the Disease Vector Culex quinquefasciatus: What Makes an Enzyme a Good Insecticide Sponge? - Hopkins_2017_Biochemistry_56_5512
Author(s) : Hopkins DH , Fraser NJ , Mabbitt PD , Carr PD , Oakeshott JG , Jackson CJ
Ref : Biochemistry , 56 :5512 , 2017
Abstract : Carboxylesterase (CBE)-mediated metabolic resistance to organophosphate and carbamate insecticides is a major problem for the control of insect disease vectors, such as the mosquito. The most common mechanism involves overexpression of CBEs that bind to the insecticide with high affinity, thereby sequestering them before they can interact with their target. However, the absence of any structure for an insecticide-sequestering CBE limits our understanding of the molecular basis for this process. We present the first structure of a CBE involved in sequestration, Cqestbeta2(1), from the mosquito disease vector Culex quinquefasciatus. Lysine methylation was used to obtain the crystal structure of Cqestbeta2(1), which adopts a canonical alpha/beta-hydrolase fold that has high similarity to the target of organophosphate and carbamate insecticides, acetylcholinesterase. Sequence similarity networks of the insect carboxyl/cholinesterase family demonstrate that CBEs associated with metabolic insecticide resistance across many species share a level of similarity that distinguishes them from a variety of other classes. This is further emphasized by the structural similarities and differences in the binding pocket and active site residues of Cqestbeta2(1) and other insect carboxyl/cholinesterases. Stopped-flow and steady-state inhibition studies support a major role for Cqestbeta2(1) in organophosphate resistance and a minor role in carbamate resistance. Comparison with another isoform associated with insecticide resistance, Cqestbeta1, showed both enzymes have similar affinity to insecticides, despite 16 amino acid differences between the two proteins. This provides a molecular understanding of pesticide sequestration by insect CBEs and could facilitate the design of CBE-specific inhibitors to circumvent this resistance mechanism in the future.
ESTHER : Hopkins_2017_Biochemistry_56_5512
PubMedSearch : Hopkins_2017_Biochemistry_56_5512
PubMedID: 28929747
Gene_locus related to this paper: culqu-1estb

Title : The Synthesis of Certain Derivatives and Analogues of (-)- and (+)-Galanthamine and an Assessment of their Capacities to Inhibit Acetylcholine Esterase - Buckler_2017_J.Org.Chem_82_7869
Author(s) : Buckler JN , Taher ES , Fraser NJ , Willis AC , Carr PD , Jackson CJ , Banwell MG
Ref : J Org Chem , 82 :7869 , 2017
Abstract : Syntheses of certain di- and mono-oxygenated derivatives (e.g., 2 and 3, respectively) and analogues (e.g., 4, a D-ring monoseco-analogue of 2) of both the (-)- and (+)-enantiomeric forms of the alkaloid galanthamine [(-)-1] are reported. All have been assessed for their capacities to inhibit acetylcholine esterase but, in contrast to the predictions from docking studies, none bind strongly to this enzyme.
ESTHER : Buckler_2017_J.Org.Chem_82_7869
PubMedSearch : Buckler_2017_J.Org.Chem_82_7869
PubMedID: 28671462

Title : The role of protein dynamics in the evolution of new enzyme function - Campbell_2016_Nat.Chem.Biol_12_944
Author(s) : Campbell EC , Kaltenbach M , Correy GJ , Carr PD , Porebski BT , Livingstone EK , Afriat-Jurnou L , Buckle AM , Weik M , Hollfelder F , Tokuriki N , Jackson CJ
Ref : Nat Chemical Biology , 12 :944 , 2016
Abstract : Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
ESTHER : Campbell_2016_Nat.Chem.Biol_12_944
PubMedSearch : Campbell_2016_Nat.Chem.Biol_12_944
PubMedID: 27618189

Title : Mapping the Accessible Conformational Landscape of an Insect Carboxylesterase Using Conformational Ensemble Analysis and Kinetic Crystallography - Correy_2016_Structure_24_977
Author(s) : Correy GJ , Carr PD , Meirelles T , Mabbitt PD , Fraser NJ , Weik M , Jackson CJ
Ref : Structure , 24 :977 , 2016
Abstract : The proper function of enzymes often depends upon their efficient interconversion between particular conformational sub-states on a free-energy landscape. Experimentally characterizing these sub-states is challenging, which has limited our understanding of the role of protein dynamics in many enzymes. Here, we have used a combination of kinetic crystallography and detailed analysis of crystallographic protein ensembles to map the accessible conformational landscape of an insect carboxylesterase (LcalphaE7) as it traverses all steps in its catalytic cycle. LcalphaE7 is of special interest because of its evolving role in organophosphate insecticide resistance. Our results reveal that a dynamically coupled network of residues extends from the substrate-binding site to a surface loop. Interestingly, the coupling of this network that is apparent in the apoenzyme appears to be reduced in the phosphorylated enzyme intermediate. Altogether, the results of this work highlight the importance of protein dynamics to enzyme function and the evolution of new activity.
ESTHER : Correy_2016_Structure_24_977
PubMedSearch : Correy_2016_Structure_24_977
PubMedID: 27210287
Gene_locus related to this paper: luccu-E3aest7

Title : Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron - Sugrue_2015_Appl.Environ.Microbiol_81_2612
Author(s) : Sugrue E , Fraser NJ , Hopkins DH , Carr PD , Khurana JL , Oakeshott JG , Scott C , Jackson CJ
Ref : Applied Environmental Microbiology , 81 :2612 , 2015
Abstract : The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.
ESTHER : Sugrue_2015_Appl.Environ.Microbiol_81_2612
PubMedSearch : Sugrue_2015_Appl.Environ.Microbiol_81_2612
PubMedID: 25636851

Title : Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing - Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
Author(s) : Porter JL , Carr PD , Collyer CA , Ollis DL
Ref : Acta Crystallographica F Struct Biol Commun , 70 :884 , 2014
Abstract : Dienelactone hydrolase (DLH) is a monomeric protein with a simple [alpha]/[beta]-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 A 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.
ESTHER : Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
PubMedSearch : Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
PubMedID: 25005082
Gene_locus related to this paper: psepu-clcd1

Title : A 5000-fold increase in the specificity of a bacterial phosphotriesterase for malathion through combinatorial active site mutagenesis - Naqvi_2014_PLoS.One_9_e94177
Author(s) : Naqvi T , Warden AC , French N , Sugrue E , Carr PD , Jackson CJ , Scott C
Ref : PLoS ONE , 9 :e94177 , 2014
Abstract : Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in kcat/KM for malathion. X-ray crystal structures for the PTEAr Ser308Leu\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.
ESTHER : Naqvi_2014_PLoS.One_9_e94177
PubMedSearch : Naqvi_2014_PLoS.One_9_e94177
PubMedID: 24721933

Title : Structure and function of an insect alpha-carboxylesterase (alphaEsterase7) associated with insecticide resistance - Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
Author(s) : Jackson CJ , Liu JW , Carr PD , Younus F , Coppin C , Meirelles T , Lethier M , Pandey G , Ollis DL , Russell RJ , Weik M , Oakeshott JG
Ref : Proc Natl Acad Sci U S A , 110 :10177 , 2013
Abstract : Insect carboxylesterases from the alphaEsterase gene cluster, such as alphaE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcalphaE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of alpha-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcalphaE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical alpha/beta-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal alpha-helix that serves as a membrane anchor. Soaking of LcalphaE7 crystals in OPs led to the capture of a crystallographic snapshot of LcalphaE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcalphaE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants ( approximately 10(6) M(-1) s(-1)) indicative of a natural substrate.
ESTHER : Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
PubMedSearch : Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
PubMedID: 23733941
Gene_locus related to this paper: luccu-E3aest7

Title : Alpha\/beta hydrolase fold: an update - Carr_2009_Protein.Pept.Lett_16_1137
Author(s) : Carr PD , Ollis DL
Ref : Protein Pept Lett , 16 :1137 , 2009
Abstract : The alpha/beta hydrolase superfamily has rapidly expanded in recent years and continues to do so at an expeditious pace. According to the ESTHER database (http://bioweb.ensam.inra.fr/ESTHER) 29000 papers have been published cataloguing 89 family groups, comprising a total of 15438 gene loci and 666 structures. This paper presents a snapshot of the current family taxonomy, catalytic chemistries, structural topologies and useful technologies emerging from the knowledge base at the current time.
ESTHER : Carr_2009_Protein.Pept.Lett_16_1137
PubMedSearch : Carr_2009_Protein.Pept.Lett_16_1137
PubMedID: 19508187

Title : In crystallo capture of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase - Jackson_2008_J.Mol.Biol_375_1189
Author(s) : Jackson CJ , Foo JL , Kim HK , Carr PD , Liu JW , Salem G , Ollis DL
Ref : Journal of Molecular Biology , 375 :1189 , 2008
Abstract : The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The mu-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that mu-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favour attack from a solvent molecule terminally coordinated to the alpha-metal ion. The bridging of both metal ions by the product, without disruption of the mu-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the beta-metal ion, displacing the mu-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a mu-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK(a) for the nucleophile, consistent with a general base function during catalysis.
ESTHER : Jackson_2008_J.Mol.Biol_375_1189
PubMedSearch : Jackson_2008_J.Mol.Biol_375_1189
PubMedID: 18082180

Title : Following directed evolution with crystallography: structural changes observed in changing the substrate specificity of dienelactone hydrolase - Kim_2005_Acta.Crystallogr.D.Biol.Crystallogr_61_920
Author(s) : Kim HK , Liu JW , Carr PD , Ollis DL
Ref : Acta Crystallographica D Biol Crystallogr , 61 :920 , 2005
Abstract : The enzyme dienelactone hydrolase (DLH) has undergone directed evolution to produce a series of mutant proteins that have enhanced activity towards the non-physiological substrates alpha-naphthyl acetate and p-nitrophenyl acetate. In terms of steady-state kinetics, the mutations caused a drop in the K(m) for the hydrolysis reaction with these two substrates. For the best mutant, there was a 5.6-fold increase in k(cat)/K(m) for the hydrolysis of alpha-naphthyl acetate and a 3.6-fold increase was observed for p-nitrophenyl acetate. For alpha-naphthyl acetate the pre-steady-state kinetics revealed that the rate constant for the formation of the covalent intermediate had increased. The mutations responsible for the rate enhancements map to the active site. The structures of the starting and mutated proteins revealed small changes in the protein owing to the mutations, while the structures of the same proteins with an inhibitor co-crystallized in the active site indicated that the mutations caused significant changes in the way the mutated proteins recognized the substrates. Within the active site of the mutant proteins, the inhibitor was rotated by about 180 degrees with respect to the orientation found in the starting enzyme. This rotation of the inhibitor caused the displacement of a large section of a loop on one side of the active site. Residues that could stabilize the transition state for the reaction were identified.
ESTHER : Kim_2005_Acta.Crystallogr.D.Biol.Crystallogr_61_920
PubMedSearch : Kim_2005_Acta.Crystallogr.D.Biol.Crystallogr_61_920
PubMedID: 15983415
Gene_locus related to this paper: psepu-clcd1

Title : Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution - Yang_2003_Protein.Eng_16_135
Author(s) : Yang H , Carr PD , McLoughlin SY , Liu JW , Horne I , Qiu X , Jeffries CM , Russell RJ , Oakeshott JG , Ollis DL
Ref : Protein Engineering , 16 :135 , 2003
Abstract : Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.
ESTHER : Yang_2003_Protein.Eng_16_135
PubMedSearch : Yang_2003_Protein.Eng_16_135
PubMedID: 12676982

Title : Expression, purification and preliminary crystallographic studies of a hyperthermophilic esterase from Archaeoglobus fulgidus - Liu_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_900
Author(s) : Liu JW , Verger D , Carr PD , Yang H , Ollis DL
Ref : Acta Crystallographica D Biol Crystallogr , 56 :900 , 2000
Abstract : An esterase from the hyperthermophilic archeon Archaeoglobus fulgidus has been expressed, purified and crystallized in a form suitable for structure analysis. The enzyme has a molecular mass of 35 467 Da and shows sequence similarity to other esterases known to possess the alpha/beta hydrolase fold. The crystals diffract to 2.8 A and belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 155.6, b = 155.0, c = 162.4 A.
ESTHER : Liu_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_900
PubMedSearch : Liu_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_900
PubMedID: 10930838

Title : Structure of the C123S mutant of dienelactone hydrolase (DLH) bound with the PMS moiety of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) - Robinson_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1376
Author(s) : Robinson A , Edwards KJ , Carr PD , Barton JD , Ewart GD , Ollis DL
Ref : Acta Crystallographica D Biol Crystallogr , 56 :1376 , 2000
Abstract : The structure of DLH (C123S) with PMS bound was solved to 2.5 A resolution (R factor = 15.1%). PMSF in 2-propanol was delivered directly to crystals in drops and unexpectedly caused the crystals to dissolve. New crystals displaying a different morphology emerged within 2 h in situ, a phenomenon that appears to be described for the first time. The changed crystal form reflected altered crystal-packing arrangements elicited by structural changes to the DLH (C123S) molecule on binding inhibitor. The new unit cell remained in the P2(1)2(1)2(1) space group but possessed different dimensions. The structure showed that PMS binding in DLH (C123S) caused conformational changes in the active site and in four regions of the polypeptide chain that contain reverse turns. In the active site, residues with aromatic side chains were repositioned in an edge-to-face cluster around the PMS phenyl ring. Their redistribution prevented restabilization of the triad His202 side chain, which was disordered in electron-density maps. Movements of other residues in the active site were shown to be related to the four displaced regions of the polypeptide chain. Their implied synergy suggests that DLH may be able to accommodate and catalyse a range of compounds unrelated to the natural substrate owing to an inherent coordinated flexibility in its overall structure. Implications for mechanism and further engineering studies are discussed.
ESTHER : Robinson_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1376
PubMedSearch : Robinson_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1376
PubMedID: 11053834
Gene_locus related to this paper: psepu-clcd1