Liu JW

References (10)

Title : Enzymatic reaction modulated DNA assembly on graphitic carbon nitride nanosheets for sensitive fluorescence detection of acetylcholinesterase activity and inhibition - Chen_2023_Mikrochim.Acta_190_268
Author(s) : Chen T , Qin Y , Wang B , Lai R , Tan G , Liu JW
Ref : Mikrochim Acta , 190 :268 , 2023
Abstract : A novel fluorescent strategy has been developed by using an enzymatic reaction modulated DNA assembly on graphitic carbon nitride nanosheets (CNNS) for the detection of acetylcholinesterase (AChE) activity and its inhibitors. The two-dimensional and ultrathin-layer CNNS-material was successfully synthesized through a chemical oxidation and ultrasound exfoliation method. Because of its excellent adsorption selectivity to ssDNA over dsDNA and superior quenching ability toward the fluorophore labels, CNNS were employed to construct a sensitive fluorescence sensing platform for the detection of AChE activity and inhibition. The detection was based on enzymatic reaction modulated DNA assembly on CNNS, which involved the specific AChE-catalyzed reaction-mediated DNA/Hg(2+) conformational change and subsequent signal transduction and amplification via hybridization chain reaction (HCR). Under the excitation at 485 nm, the fluorescence signal from 500 to 650 nm (lambda(max) = 518 nm) of the developed sensing system was gradually increased with increasing concentration of AChE. The quantitative determination range of AChE is from 0.02 to 1 mU/mL and the detection limit was 0.006 mU/mL. The developed strategy was successfully applied to the assay of AChE in human serum samples, and can also be used to effectively screen AChE inhibitors, showing great promise providing a robust and effective platform for AChE-related diagnosis, drug screening, and therapy.
ESTHER : Chen_2023_Mikrochim.Acta_190_268
PubMedSearch : Chen_2023_Mikrochim.Acta_190_268
PubMedID: 37338607

Title : Improving on nature's shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability - Alfaro-Chavez_2019_Protein.Eng.Des.Sel_32_13
Author(s) : Alfaro-Chavez AL , Liu JW , Porter JL , Goldman A , Ollis DL
Ref : Protein Engineering Des Sel , 32 :13 , 2019
Abstract : An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37 degrees C, while the mutant with P291L of 42.2 degrees C and the mutant R7_47D with five mutations had a value of 52.9 degrees C, corresponding to an improvement of 16 degrees C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.
ESTHER : Alfaro-Chavez_2019_Protein.Eng.Des.Sel_32_13
PubMedSearch : Alfaro-Chavez_2019_Protein.Eng.Des.Sel_32_13
PubMedID: 31403166
Gene_locus related to this paper: drome-lip3

Title : Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila - Younus_2017_Sci.Rep_7_46188
Author(s) : Younus F , Fraser NJ , Coppin CW , Liu JW , Correy GJ , Chertemps T , Pandey G , Maibeche M , Jackson CJ , Oakeshott JG
Ref : Sci Rep , 7 :46188 , 2017
Abstract : Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate.
ESTHER : Younus_2017_Sci.Rep_7_46188
PubMedSearch : Younus_2017_Sci.Rep_7_46188
PubMedID: 28393888
Gene_locus related to this paper: drome-este6

Title : Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability - Fraser_2016_J.Mol.Biol_428_2359
Author(s) : Fraser NJ , Liu JW , Mabbitt PD , Correy GJ , Coppin CW , Lethier M , Perugini MA , Murphy JM , Oakeshott JG , Weik M , Jackson CJ
Ref : Journal of Molecular Biology , 428 :2359 , 2016
Abstract : Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the alphaE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcalphaE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcalphaE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.
ESTHER : Fraser_2016_J.Mol.Biol_428_2359
PubMedSearch : Fraser_2016_J.Mol.Biol_428_2359
PubMedID: 27016206
Gene_locus related to this paper: luccu-E3aest7

Title : Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera - Li_2013_PLoS.One_8_e77685
Author(s) : Li Y , Farnsworth CA , Coppin CW , Teese MG , Liu JW , Scott C , Zhang X , Russell RJ , Oakeshott JG
Ref : PLoS ONE , 8 :e77685 , 2013
Abstract : Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.
ESTHER : Li_2013_PLoS.One_8_e77685
PubMedSearch : Li_2013_PLoS.One_8_e77685
PubMedID: 24204917
Gene_locus related to this paper: helam-d5g3c9 , helam-d5g3d2 , helam-d5g3d3 , helam-d5g3d4 , helam-d5g3d5 , helam-d5kx87 , helam-d5kx99 , helam-d5kxa9 , helam-d9iv61 , helam-d9iv62 , helam-h9zvh4 , helam-s4wfz6

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 : 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. 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