Ollis DL

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Full name : Ollis David L

First name : David L

Mail : Research School of Chemistry, Australian National University, Australian Capital Territory 0200

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Country : Australia

Email : ollis@rsc.anu.edu.au

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References (26)

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 : Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search - Porter_2015_ACS.Chem.Biol_10_611
Author(s) : Porter JL , Boon PL , Murray TP , Huber T , Collyer CA , Ollis DL
Ref : ACS Chemical Biology , 10 :611 , 2015
Abstract : The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse alpha/beta hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
ESTHER : Porter_2015_ACS.Chem.Biol_10_611
PubMedSearch : Porter_2015_ACS.Chem.Biol_10_611
PubMedID: 25419863
Gene_locus related to this paper: psepu-clcd1

Title : Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity - Porter_2015_Protein.J_34_82
Author(s) : Porter JL , Collyer CA , Ollis DL
Ref : Protein J , 34 :82 , 2015
Abstract : Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.
ESTHER : Porter_2015_Protein.J_34_82
PubMedSearch : Porter_2015_Protein.J_34_82
PubMedID: 25600287

Title : Altering the substrate specificity of methyl parathion hydrolase with directed evolution - Ng_2015_Arch.Biochem.Biophys_573_59
Author(s) : Ng TK , Gahan LR , Schenk G , Ollis DL
Ref : Archives of Biochemistry & Biophysics , 573 :59 , 2015
Abstract : Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1x106s-1M-1), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1x103s-1M-1). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9x105s-1M-1). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.
ESTHER : Ng_2015_Arch.Biochem.Biophys_573_59
PubMedSearch : Ng_2015_Arch.Biochem.Biophys_573_59
PubMedID: 25797441

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 : Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH) - Pedroso_2014_J.Biol.Inorg.Chem_19_1263
Author(s) : Pedroso MM , Ely F , Mitic N , Carpenter MC , Gahan LR , Wilcox DE , Larrabee JL , Ollis DL , Schenk G
Ref : J Biol Inorg Chem , 19 :1263 , 2014
Abstract : Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90s% identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.
ESTHER : Pedroso_2014_J.Biol.Inorg.Chem_19_1263
PubMedSearch : Pedroso_2014_J.Biol.Inorg.Chem_19_1263
PubMedID: 25104333

Title : Use of OpdA, an organophosphorus (OP) hydrolase, prevents lethality in an African green monkey model of acute OP poisoning - Jackson_2014_Toxicology_317_1
Author(s) : Jackson CJ , Carville A , Ward J , Mansfield K , Ollis DL , Khurana T , Bird SB
Ref : Toxicology , 317 :1 , 2014
Abstract : Organophosphorus (OP) pesticides are a diverse class of acetylcholinesterase (AChE) inhibitors that are responsible for tremendous morbidity and mortality worldwide, killing approximately 300,000 people annually. Enzymatic hydrolysis of OPs is a potential therapy for acute poisoning. OpdA, an OP hydrolase isolated from Agrobacterium radiobacter, has been shown to decrease lethality in rodent models of OP poisoning. This study investigated the effects of OpdA on AChE activity, plasma concentrations of OP, and signs of toxicity after administration of dichlorvos to nonhuman primates. A dose of 75mg/kg dichlorvos given orally caused apnea within 10min with a progressive decrease in heart rate. Blood AChE activity decreased to zero within 10min. Respirations and AChE activity did not recover. The mean dichlorvos concentration rose to a peak of 0.66mug/ml. Treated monkeys received 1.2mg/kg OpdA iv immediately after poisoning with dichlorvos. In Opda-treated animals, heart and respiratory rates were unchanged from baseline over a 240-minute observation period. AChE activity slowly declined, but remained above 25% of baseline for the entire duration. Dichlorvos concentrations reached a mean peak of 0.19mug/ml at 40min after poisoning and decreased to a mean of 0.05mug/ml at 240min. These results show that OpdA hydrolyzes dichlorvos in an African green monkey model of lethal poisoning, delays AChE inhibition, and prevents lethality.
ESTHER : Jackson_2014_Toxicology_317_1
PubMedSearch : Jackson_2014_Toxicology_317_1
PubMedID: 24447378

Title : Promiscuity comes at a price: catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ - Daumann_2013_Biochim.Biophys.Acta_1834_425
Author(s) : Daumann LJ , McCarthy BY , Hadler KS , Murray TP , Gahan LR , Larrabee JA , Ollis DL , Schenk G
Ref : Biochimica & Biophysica Acta , 1834 :425 , 2013
Abstract : The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) is a highly promiscuous dinuclear metallohydrolase with respect to both substrate specificity and metal ion composition. While this promiscuity may adversely affect the enzyme's catalytic efficiency its ability to hydrolyse some organophosphates (OPs) and by-products of OP degradation have turned GpdQ into a promising candidate for bioremedial applications. Here, we investigated both metal ion binding and the effect of the metal ion composition on catalysis. The prevalent in vivo metal ion composition for GpdQ is proposed to be of the type Fe(II)Zn(II), a reflection of natural abundance rather than catalytic optimisation. The Fe(II) appears to have lower binding affinity than other divalent metal ions, and the catalytic efficiency of this mixed metal center is considerably smaller than that of Mn(II), Co(II) or Cd(II)-containing derivatives of GpdQ. Interestingly, metal ion replacements do not only affect catalytic efficiency but also the optimal pH range for the reaction, suggesting that different metal ion combinations may employ different mechanistic strategies. These metal ion-triggered modulations are likely to be mediated via an extensive hydrogen bond network that links the two metal ion binding sites via residues in the substrate binding pocket. The observed functional diversity may be the cause for the modest catalytic efficiency of wild-type GpdQ but may also be essential to enable the enzyme to evolve rapidly to alter substrate specificity and enhance k(cat) values, as has recently been demonstrated in a directed evolution experiment. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.
ESTHER : Daumann_2013_Biochim.Biophys.Acta_1834_425
PubMedSearch : Daumann_2013_Biochim.Biophys.Acta_1834_425
PubMedID: 22366468

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 : Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter - Ely_2012_J.Inorg.Biochem_106_19
Author(s) : Ely F , Pedroso MM , Gahan LR , Ollis DL , Guddat LW , Schenk G
Ref : J Inorg Biochem , 106 :19 , 2012
Abstract : OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 A resolution. The structure shows the phosphate bound in a tripodal mode to the metal ions whereby two of the oxygen atoms of PO(4) are terminally bound to each metal ion and a third oxygen bridges the two metal ions, thus displacing the muOH in the active site. In silico modelling demonstrates that the phosphate moiety of a reaction product, e.g. diethyl phosphate, may bind in the same orientation, positioning the diethyl groups neatly into the substrate binding pocket close to the metal center. Thus, similar to the binuclear metallohydrolases urease and purple acid phosphatase the tripodal arrangement of PO(4) is interpreted in terms of a role of the muOH as a reaction nucleophile.
ESTHER : Ely_2012_J.Inorg.Biochem_106_19
PubMedSearch : Ely_2012_J.Inorg.Biochem_106_19
PubMedID: 22112835

Title : Binuclear metallohydrolases: complex mechanistic strategies for a simple chemical reaction - Schenk_2012_Acc.Chem.Res_45_1593
Author(s) : Schenk G , Mitic N , Gahan LR , Ollis DL , McGeary RP , Guddat LW
Ref : Acc Chem Res , 45 :1593 , 2012
Abstract : Binuclear metallohydrolases are a large family of enzymes that require two closely spaced transition metal ions to carry out a plethora of hydrolytic reactions. Representatives include purple acid phosphatases (PAPs), enzymes that play a role in bone metabolism and are the only member of this family with a heterovalent binuclear center in the active form (Fe(3+)-M(2+), M = Fe, Zn, Mn). Other members of this family are urease, which contains a di-Ni(2+) center and catalyzes the breakdown of urea, arginase, which contains a di-Mn(2+) center and catalyzes the final step in the urea cycle, and the metallo-beta-lactamases, which contain a di-Zn(2+) center and are virulence factors contributing to the spread of antibiotic-resistant pathogens. Binuclear metallohydrolases catalyze numerous vital reactions and are potential targets of drugs against a wide variety of human disorders including osteoporosis, various cancers, antibiotic resistance, and erectile dysfunctions. These enzymes also tend to catalyze more than one reaction. An example is an organophosphate (OP)-degrading enzyme from Enterobacter aerogenes (GpdQ). Although GpdQ is part of a pathway that is used by bacteria to degrade glycerolphosphoesters, it hydrolyzes a variety of other phosphodiesters and displays low levels of activity against phosphomono- and triesters. Such a promiscuous nature may have assisted the apparent recent evolution of some binuclear metallohydrolases to deal with situations created by human intervention such as OP pesticides in the environment. OP pesticides were first used approximately 70 years ago, and therefore the enzymes that bacteria use to degrade them must have evolved very quickly on the evolutionary time scale. The promiscuous nature of enzymes such as GpdQ makes them ideal candidates for the application of directed evolution to produce new enzymes that can be used in bioremediation and against chemical warfare. In this Account, we review the mechanisms employed by binuclear metallohydrolases and use PAP, the OP-degrading enzyme from Agrobacterium radiobacter (OPDA), and GpdQ as representative systems because they illustrate both the diversity and similarity of the reactions catalyzed by this family of enzymes. The majority of binuclear metallohydrolases utilize metal ion-activated water molecules as nucleophiles to initiate hydrolysis, while some, such as alkaline phosphatase, employ an intrinsic polar amino acid. Here we only focus on catalytic strategies applied by the former group.
ESTHER : Schenk_2012_Acc.Chem.Res_45_1593
PubMedSearch : Schenk_2012_Acc.Chem.Res_45_1593
PubMedID: 22698580

Title : Substrate-induced conformational change and isomerase activity of dienelactone hydrolase and its site-specific mutants - Walker_2012_Chembiochem_13_1645
Author(s) : Walker I , Hennessy JE , Ollis DL , Easton CJ
Ref : Chembiochem , 13 :1645 , 2012
Abstract : Studies of the interactions of dienelactone hydrolase (DLH) and its mutants with both E and Z dienelactone substrates show that the enzyme exhibits two different conformational responses specific for hydrolysis of each of its substrate isomers. DLH facilitates hydrolysis of the Z dienelactone through an unusual charge-relay system that is initiated by interaction between the substrate carboxylate and an enzyme arginine residue that activates an otherwise non-nucleophilic cysteine. The E dienelactone does not display this substrate-arginine binding interaction, but instead induces an alternate conformational response that promotes hydrolysis. Furthermore, the substitution of cysteine 123 for serine (C123S) in DLH, instead of inactivating the enzyme as is typical for this active-site mutation, changes the catalysis from substrate hydrolysis to isomerisation. This is due to the deacylation of the acyl-enzyme intermediates being much slower, thereby increasing their lifetimes and allowing for their interconversion through isomerisation, followed by relactonisation.
ESTHER : Walker_2012_Chembiochem_13_1645
PubMedSearch : Walker_2012_Chembiochem_13_1645
PubMedID: 22761053

Title : Electronic and geometric structures of the organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) - Ely_2011_J.Biol.Inorg.Chem_16_777
Author(s) : Ely F , Hadler KS , Mitic N , Gahan LR , Ollis DL , Plugis NM , Russo MT , Larrabee JA , Schenk G
Ref : J Biol Inorg Chem , 16 :777 , 2011
Abstract : The organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) is a highly efficient catalyst for the degradation of pesticides and some nerve agents such as sarin. OpdA requires two metal ions for catalytic activity, and hydrolysis is initiated by a nucleophilic hydroxide that is bound to one of these metal ions. The precise location of this nucleophile has been contentious, with both a terminal and a metal-ion-bridging hydroxide as likely candidates. Here, we employed magnetic circular dichroism to probe the electronic and geometric structures of the Co(II)-reconstituted dinuclear metal center in OpdA. In the resting state the metal ion in the more secluded alpha site is five-coordinate, whereas the Co(II) in the solvent-exposed beta site is predominantly six-coordinate with two terminal water ligands. Addition of the slow substrate diethyl 4-methoxyphenyl phosphate does not affect the alpha site greatly but lowers the coordination number of the beta site to five. A reduction in the exchange coupling constant indicates that substrate binding also triggers a shift of the mu-hydroxide into a pseudoterminal position in the coordination sphere of either the alpha or the beta metal ion. Mechanistic implications of these observations are discussed.
ESTHER : Ely_2011_J.Biol.Inorg.Chem_16_777
PubMedSearch : Ely_2011_J.Biol.Inorg.Chem_16_777
PubMedID: 21487938

Title : Pharmacokinetics of OpdA, an organophosphorus hydrolase, in the African green monkey - Jackson_2010_Biochem.Pharmacol_80_1075
Author(s) : Jackson CJ , Scott C , Carville A , Mansfield K , Ollis DL , Bird SB
Ref : Biochemical Pharmacology , 80 :1075 , 2010
Abstract : 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 are as high as 40%. Enzymatic hydrolysis of OPs is an attractive new potential therapy for acute OP poisoning. A number of bacterial OP hydrolases have been isolated. A promising OP hydrolase is an enzyme isolated from Agrobacterium radiobacter, named OpdA. OpdA has been shown to decrease lethality in rodent models of parathion and dichlorvos poisoning. However, pharmacokinetic data have not been obtained. In this study, we examined the pharmacokinetics of OpdA in an African Green Monkey model. At a dose of 1.2mg/kg the half-life of OpdA was approximately 40 min, with a mean residence time of 57 min. As expected, the half-life did not change with the dose of OpdA given: at doses of 0.15 and 0.45 mg/kg, the half-life of OpdA was 43.1 and 38.9 min, respectively. In animals subjected to 5 daily doses of OpdA, the residual activity that was measured 24h after each OpdA dose increased 5-fold for the 0.45 mg/kg dose and 11-fold for the 1.2mg/kg dose. OpdA exhibits pharmacokinetics favorable for the further development as a therapy for acute OP poisoning, particularly for hydrophilic OP pesticides. Future work to increase the half-life of OpdA may be beneficial.
ESTHER : Jackson_2010_Biochem.Pharmacol_80_1075
PubMedSearch : Jackson_2010_Biochem.Pharmacol_80_1075
PubMedID: 20599794

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 : Comparing the organophosphorus and carbamate insecticide resistance mutations in cholin- and carboxyl-esterases - Oakeshott_2005_Chem.Biol.Interact_157-158_269
Author(s) : Oakeshott JG , Devonshire AL , Claudianos C , Sutherland TD , Horne I , Campbell PM , Ollis DL , Russell RJ
Ref : Chemico-Biological Interactions , 157-158 :269 , 2005
Abstract : Mutant insect carboxyl/cholinesterases underlie over 60 cases of resistance to organophosphorus and/or carbamate insecticides. Biochemical and molecular data on about 20 of these show recurrent use of a very small number of mutational options to generate either target site or metabolic resistance. Moreover, the mutant enzymes are often kinetically inefficient and associated with significant fitness costs, due to impaired performance of the enzymes' original function. By contrast many bacterial enzymes are now known which can effectively detoxify these pesticides. It appears that the constraints of the genetic code and eukaryote genetic systems have severely limited the evolutionary response of insects to the widespread use of the insecticides over the last 60 years.
ESTHER : Oakeshott_2005_Chem.Biol.Interact_157-158_269
PubMedSearch : Oakeshott_2005_Chem.Biol.Interact_157-158_269
PubMedID: 16289012

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 : The role of inhibition in enzyme evolution -
Author(s) : McLoughlin SY , Ollis DL
Ref : Chemical Biology , 11 :735 , 2004
PubMedID: 15217603

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

Title : Of barn owls and bankers: a lush variety of alpha\/beta hydrolases - Heikinheimo_1999_Structure.Fold.Des_7_R141
Author(s) : Heikinheimo P , Goldman A , Jeffries C , Ollis DL
Ref : Structure Fold Des , 7 :R141 , 1999
Abstract : alpha/beta Hydrolase fold proteins are an important, diverse, widespread group of enzymes not yet fully exploited by structural biologists. We describe the current state of knowledge of this family, and suggest a smaller definition of the required core and some possible future avenues of exploration.
ESTHER : Heikinheimo_1999_Structure.Fold.Des_7_R141
PubMedSearch : Heikinheimo_1999_Structure.Fold.Des_7_R141
PubMedID: 10404588

Title : A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly - Newcomb_1997_Proc.Natl.Acad.Sci.U.S.A_94_7464
Author(s) : Newcomb RD , Campbell PM , Ollis DL , Cheah E , Russell RJ , Oakeshott JG
Ref : Proceedings of the National Academy of Sciences of the United States of America , 94 :7464 , 1997
Abstract : Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.
ESTHER : Newcomb_1997_Proc.Natl.Acad.Sci.U.S.A_94_7464
PubMedSearch : Newcomb_1997_Proc.Natl.Acad.Sci.U.S.A_94_7464
PubMedID: 9207114
Gene_locus related to this paper: luccu-ACHE , luccu-E3aest7

Title : The alpha\/beta hydrolase fold - Ollis_1992_Prot.Engin_5_197
Author(s) : Ollis DL , Cheah E , Cygler M , Dijkstra B , Frolow F , Franken SM , Harel M , Remington SJ , Silman I , Schrag JD , Sussman JL , Verschueren KHG , Goldman A
Ref : Protein Engineering , 5 :197 , 1992
Abstract : We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
ESTHER : Ollis_1992_Prot.Engin_5_197
PubMedSearch : Ollis_1992_Prot.Engin_5_197
PubMedID: 1409539

Title : Crystallization and preliminary x-ray crystallographic data of dienelactone hydrolase from Pseudomonas sp. B13 - Ollis_1985_J.Biol.Chem_260_9818
Author(s) : Ollis DL , Ngai KL
Ref : Journal of Biological Chemistry , 260 :9818 , 1985
Abstract : Dienelactone hydrolase (EC 3.1.1.45) from Pseudomonas sp. B13 has been crystallized in a form suitable for high resolution x-ray diffraction study. The crystals are orthorhombic, the space group being P212121, with unit cell dimensions a = 48.9 A, b = 71.2 A, and c = 77.5 A. There appears to be 1 molecule in the asymmetric unit.
ESTHER : Ollis_1985_J.Biol.Chem_260_9818
PubMedSearch : Ollis_1985_J.Biol.Chem_260_9818
PubMedID: 4019496
Gene_locus related to this paper: psepu-clcd1