Jackson CJ

General

Full name : Jackson Colin J

First name : Colin J

Mail : Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.

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Country : Australia

Email : colin.jackson@anu.edu.au

Phone : +33646749765

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References (25)

Title : Improving plastic degrading enzymes via directed evolution - Joho_2024_Protein.Eng.Des.Sel_37_
Author(s) : Joho Y , Vongsouthi V , Gomez C , Larsen JS , Ardevol A , Jackson CJ
Ref : Protein Engineering Des Sel , 37 : , 2024
Abstract : Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.
ESTHER : Joho_2024_Protein.Eng.Des.Sel_37_
PubMedSearch : Joho_2024_Protein.Eng.Des.Sel_37_
PubMedID: 38713696

Title : Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity - Joho_2022_Biochemistry__
Author(s) : Joho Y , Vongsouthi V , Spence MA , Ton J , Gomez C , Tan LL , Kaczmarski JA , Caputo AT , Royan S , Jackson CJ , Ardevol A
Ref : Biochemistry , : , 2022
Abstract : The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 degreesC higher than that of IsPETase and two variants with significantly higher catalytic activity.
ESTHER : Joho_2022_Biochemistry__
PubMedSearch : Joho_2022_Biochemistry__
PubMedID: 35951410
Gene_locus related to this paper: idesa-peth

Title : Dan Salah Tawfik (1955-2021) A giant of protein evolution - Elias_2021_EMBO.Rep_22_e53256
Author(s) : Elias MH , Fraser JS , Kamerlin SCL , Patrick WM , Jackson CJ
Ref : EMBO Rep , 22 :e53256 , 2021
Abstract : It was with great sorrow that we have learned of the untimely death of our friend, mentor, collaborator, and hero, Dan Tawfik. Danny was a true legend in the field of protein function and evolution. He had an incredibly creative mind and a breadth of knowledge - his interests spanned chemistry and engineering to genetics and evolution - that allowed him to see connections that the rest of us could not. More importantly, he made solving biochemical mysteries fun: He was passionate about his work, and his face lit up with joy whenever he talked about scientific topics that excited him (of which there were a lot). Conversations with Danny made us all smarter by osmosis. Danny's own evolution in science began with physical organic chemistry and biochemistry. His PhD at the Weizmann Institute of Science, awarded in 1995, was on catalytic antibodies under the supervision of Zelig Eshhar and Michael Sela. It was followed by a highly productive period at the University of Cambridge's Centre for Protein Engineering, first as a postdoctoral fellow with Alan Fersht and Tony Kirby, and then as a senior researcher. Among his many achievements during his time in Cambridge was the demonstration that off-the-shelf proteins - the serum albumins - could rival the best catalytic antibodies in accelerating the Kemp elimination reaction due to non-specific medium effects. This work was an early example of unexpected catalytic promiscuity, and it sowed the seed for Danny's later fascination with -Y'esoteric, niche enzymology' that went far beyond convenient model systems. It was also in Cambridge where Danny first realized the power of the then new field of directed evolution, both for biotechnology and for elucidating evolutionary processes. He and Andrew Griffiths pioneered emulsion-based in vitro compartmentalization. The idea of controlling biochemical reactions in separate aqueous droplets inspired emulsion PCR and next-generation sequencing technologies, whereas Danny used it to solve a long-standing problem in directed evolution; in vitro selection techniques had always been good at identifying ligand-binding proteins, but compartmentalization finally enabled the directed evolution of ultra-fast catalysts. Danny returned to Israel in 2001 to join the faculty of the Weizmann Institute of Science where his scientific trajectory further evolved, diverged, and even 'drifted'. He developed new methods for enzyme engineering and applied his evolutionary insights into de novo protein design efforts. In this context, Danny's interest was always focused on how proteins evolve, particularly the connection between promiscuity, conformational diversity, and evolvability. His depth of understanding underpinned both applied research, such as engineering enzymes to detoxify nerve agents, and fundamental research, such as the evolution of enzymes from non-catalytic scaffolds. Through it all, Danny retained his sense of joy and wonder at the -Y'beautiful aspects of Nature's chemistry-Y'. This includes his discovery of an exquisite molecular specificity mechanism mediated by a single, short H-bond that enables microbes to scavenge phosphate in arsenate-rich environments. In recent years, he deciphered the biosynthetic mechanism of dimethyl sulfide, 'the smell of the sea', and homed in on the interplay between the evolution of an enzyme, its host organism, and environmental complexity. His insights into how the first proteins emerged caused tremendous excitement in the field. He established the roots of two common enzyme lineages, the Rossmann and P-loop NTPases, as simple polypeptides, and suggested ornithine as the first cationic amino acid. Prior to his death, he published the results of another tour de force: evidence that the first organisms to utilize oxygen may have appeared much earlier than thought. His work impacted many research fields, and he won many significant awards. Most recently, Danny was awarded the EMET Prize for Art, Science and Culture (2020), informally dubbed 'Israel's Nobel Prize'. He was an active and valued member of the EMBO community, having been elected in 2009, and, until his passing, served on the Editorial Advisory Board of EMBO Reports. Danny was also a superb science communicator. Both his research articles and reviews are a joy to read. What stood out just as much as his brilliance was his personality, as he embodied the Yiddish concept of being a true 'mensch'. Danny was humble, was down-to-earth, and treated all his colleagues - including the most junior members of our research teams - as equals. He championed the careers of others, both those who worked directly for him and those who were lucky enough to be 'just' his friends and collaborators. He believed in us even when we did not believe in ourselves, and he was always there to answer questions both scientific and professional. While he loved to share his own ideas, he would be just as excited about ours. Despite his own busy schedule, he always found the time to help others. He was also excellent company, with a great, very dry, sense of humor, and endless interesting stories, including from his own colorful life. In the days after his untimely death, an often-repeated phrase was 'he was my best friend'. Danny's former group members have gone on to be highly successful in both industry and academia, including more than 15 former doctoral and postdoctoral researchers who are now faculty. The network of researchers Danny has trained, mentored, or influenced is broad, and this legacy is testament to his qualities as both a scientist and a person.
ESTHER : Elias_2021_EMBO.Rep_22_e53256
PubMedSearch : Elias_2021_EMBO.Rep_22_e53256
PubMedID:

Title : Overcoming insecticide resistance through computational inhibitor design - Correy_2019_Proc.Natl.Acad.Sci.U.S.A_116_21012
Author(s) : Correy GJ , Zaidman D , Harmelin A , Carvalho S , Mabbitt PD , Calaora V , James PJ , Kotze AC , Jackson CJ , London N
Ref : Proc Natl Acad Sci U S A , 116 :21012 , 2019
Abstract : Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase alphaE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp alphaE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.
ESTHER : Correy_2019_Proc.Natl.Acad.Sci.U.S.A_116_21012
PubMedSearch : Correy_2019_Proc.Natl.Acad.Sci.U.S.A_116_21012
PubMedID: 31575743
Gene_locus related to this paper: luccu-E3aest7

Title : The molecular basis for the neofunctionalization of the juvenile hormone esterase duplication in Drosophila - Hopkins_2019_Insect.Biochem.Mol.Biol_106_10
Author(s) : Hopkins DH , Rane RV , Younus F , Coppin CW , Pandey G , Jackson CJ , Oakeshott JG
Ref : Insect Biochemistry & Molecular Biology , 106 :10 , 2019
Abstract : The Drosophila melanogaster enzymes juvenile hormone esterase (DmJHE) and its duplicate, DmJHEdup, present ideal examples for studying the structural changes involved in the neofunctionalization of enzyme duplicates. DmJHE is a hormone esterase with precise regulation and highly specific activity for its substrate, juvenile hormone. DmJHEdup is an odorant degrading esterase (ODE) responsible for processing various kairomones in antennae. Our phylogenetic analysis shows that the JHE lineage predates the hemi/holometabolan split and that several duplications of JHEs have been templates for the evolution of secreted beta-esterases such as ODEs through the course of insect evolution. Our biochemical comparisons further show that DmJHE has sufficient substrate promiscuity and activity against odorant esters for a duplicate to evolve a general ODE function against a range of mid-long chain food esters, as is shown in DmJHEdup. This substrate range complements that of the only other general ODE known in this species, Esterase 6. Homology models of DmJHE and DmJHEdup enabled comparisons between each enzyme and the known structures of a lepidopteran JHE and Esterase 6. Both JHEs showed very similar active sites despite low sequence identity (30%). Both ODEs differed drastically from the JHEs and each other, explaining their complementary substrate ranges. A small number of amino acid changes are identified that may have been involved in the early stages of the neofunctionalization of DmJHEdup. Our results provide key insights into the process of neofunctionalization and the structural changes that can be involved.
ESTHER : Hopkins_2019_Insect.Biochem.Mol.Biol_106_10
PubMedSearch : Hopkins_2019_Insect.Biochem.Mol.Biol_106_10
PubMedID: 30611903
Gene_locus related to this paper: drome-CG8424 , drome-CG8425

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 : 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 : Laboratory evolution of protein conformational dynamics - Campbell_2017_Curr.Opin.Struct.Biol_50_49
Author(s) : Campbell EC , Correy GJ , Mabbitt PD , Buckle AM , Tokuriki N , Jackson CJ
Ref : Current Opinion in Structural Biology , 50 :49 , 2017
Abstract : This review focuses on recent work that has begun to establish specific functional roles for protein conformational dynamics, specifically how the conformational landscapes that proteins can sample can evolve under laboratory based evolutionary selection. We discuss recent technical advances in computational and biophysical chemistry, which have provided us with new ways to dissect evolutionary processes. Finally, we offer some perspectives on the emerging view of conformational dynamics and evolution, and the challenges that we face in rationally engineering conformational dynamics.
ESTHER : Campbell_2017_Curr.Opin.Struct.Biol_50_49
PubMedSearch : Campbell_2017_Curr.Opin.Struct.Biol_50_49
PubMedID: 29120734

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 : 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 : Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency - Mabbitt_2016_Biochemistry_55_1408
Author(s) : Mabbitt PD , Correy GJ , Meirelles T , Fraser NJ , Coote ML , Jackson CJ
Ref : Biochemistry , 55 :1408 , 2016
Abstract : The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in alpha-esterase 7 (LcalphaE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcalphaE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization.
ESTHER : Mabbitt_2016_Biochemistry_55_1408
PubMedSearch : Mabbitt_2016_Biochemistry_55_1408
PubMedID: 26881849
Gene_locus related to this paper: luccu-E3aest7

Title : Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects - Yang_2016_Biochemistry_55_4583
Author(s) : Yang G , Hong N , Baier F , Jackson CJ , Tokuriki N
Ref : Biochemistry , 55 :4583 , 2016
Abstract : How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-beta-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by -3 A through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution.
ESTHER : Yang_2016_Biochemistry_55_4583
PubMedSearch : Yang_2016_Biochemistry_55_4583
PubMedID: 27444875

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 : 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 : 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 : 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 : Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling - Afriat-Jurnou_2012_Biochemistry_51_6047
Author(s) : Afriat-Jurnou L , Jackson CJ , Tawfik DS
Ref : Biochemistry , 51 :6047 , 2012
Abstract : Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K(M) values of up to 2 x 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.
ESTHER : Afriat-Jurnou_2012_Biochemistry_51_6047
PubMedSearch : Afriat-Jurnou_2012_Biochemistry_51_6047
PubMedID: 22809311

Title : Testing the evolvability of an insect carboxylesterase for the detoxification of synthetic pyrethroid insecticides - Coppin_2012_Insect.Biochem.Mol.Biol_42_343
Author(s) : Coppin CW , Jackson CJ , Sutherland T , Hart PJ , Devonshire AL , Russell RJ , Oakeshott JG
Ref : Insect Biochemistry & Molecular Biology , 42 :343 , 2012
Abstract : Esterases have been implicated in metabolic resistance to synthetic pyrethroids in several insect species but little is yet known of the molecular basis for these effects. In this work modern directed evolution technology was used to test to what extent it is possible to genetically enhance the pyrethroid hydrolytic activity of the E3 carboxylesterase from the blowfly Lucilia cuprina. High throughput screening of a random mutant library with individual stereoisomers of fluorogenic analogues of two type II pyrethroids identified 17 promising variants that were then also tested with the commercial pyrethroid deltamethrin. Between them, these variants displayed significantly improved activities for all the substrates tested. Amino acid substitutions at ten different residues were clearly implicated in the improvements, although most only enhanced activity for a subset of the stereoisomers. Several new combinations of the most promising amino acid substitutions were then made, and negative epistatic effects were found in most of the combinations, but significant improvements were also found in a minority of them. The best mutant recovered contained three amino acid changes and hydrolysed deltamethrin at more than 100 times the rate of wild-type E3. Structural analysis shows that nine of the ten mutated residues improving pyrethroid or analogue activities cluster in putative substrate binding pockets in the active site, with the three mutations of largest effect all increasing the volume of the acyl pocket.
ESTHER : Coppin_2012_Insect.Biochem.Mol.Biol_42_343
PubMedSearch : Coppin_2012_Insect.Biochem.Mol.Biol_42_343
PubMedID: 22300675
Gene_locus related to this paper: luccu-E3aest7

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 : Characterization of the phenylurea hydrolases A and B: founding members of a novel amidohydrolase subgroup - Khurana_2009_Biochem.J_418_431
Author(s) : Khurana JL , Jackson CJ , Scott C , Pandey G , Horne I , Russell RJ , Herlt A , Easton CJ , Oakeshott JG
Ref : Biochemical Journal , 418 :431 , 2009
Abstract : Mycobacterium brisbanense strain JK1, a bacterium capable of degrading the herbicide diuron, was isolated from herbicide-exposed soil. A gene/enzyme system with diuron hydrolase activity was isolated from this strain and named PUH (phenylurea hydrolase) B (puhB/PuhB) because of its close similarity to the previously characterized PUH A (puhA/PuhA). Both PUHs were heterologously expressed, purified and characterized. The PUHs were found to oligomerize as hexamers in solution, with each monomer containing a mononuclear Zn2+ active site. Sequence analysis showed that these enzymes belong to the metal-dependent amidohydrolase superfamily, although they contain a hitherto unreported Asn-X-His metal-binding motif and appear to form a novel sub-group within this superfamily. The effects of temperature and solvent on the enzymes were characterized. Determination of the kinetic parameters of the PUHs was used alongside Bronsted plots to develop a plausible catalytic mechanism, which is similar to that used by urease. In addition to the primary PUH activity, both enzymes are catalytically promiscuous, efficiently hydrolysing esters, carbamates and phosphotriesters. In fact, an analogue of diuron, in which the C-N bond was replaced by a C-O bond, was found to be turned over as efficiently as diuron, suggesting that the substrate specificity is predominantly determined by steric factors. The discovery of PuhA and PuhB on separate continents, and the absence of any other close homologues in the available sequence databases, poses a challenging question regarding the evolutionary origins of these enzymes.
ESTHER : Khurana_2009_Biochem.J_418_431
PubMedSearch : Khurana_2009_Biochem.J_418_431
PubMedID: 19000034
Gene_locus related to this paper: 9myco-b8r4k2

Title : Use of engineered enzymes to identify organophosphate and pyrethroid-related toxicity in toxicity identification evaluations - Weston_2009_Environ.Sci.Technol_43_5514
Author(s) : Weston DP , Jackson CJ
Ref : Environ Sci Technol , 43 :5514 , 2009
Abstract : Engineered variants of a carboxylesterase from Lucilia cuprina (E3) and a phosphotriesterase from Agrobacterium radiobacter (OpdA) with enhanced hydrolytic activities against pyrethroid and organophosphate pesticides were evaluated as a toxicity identification evaluation (TIE) manipulation. Reduction in toxicity in the presence of the enzyme provides an indication that the toxicant is the enzyme's target substrate. Carboxy/esterase E3 variants were evaluated to determine if the enzymes could mitigate toxicity of pyrethroids to the amphipod, Hyalella azteca. Enzymes were able to achieve 12-70-fold reduction in toxicity for bifenthrin, cyfluthrin, and cypermethrin in water. Only a 2-fold reduction in toxicity was observed with pyrethroid-contaminated sediment though the phosphotriesterase OpdA achieved at least a 35-fold reduction in toxicity from the organophosphate chlorpyrifos in sediment. Tests with urban runoff samples and agriculture-affected sediments demonstrated that the enzymes could be useful in TIEs to identify pesticide-related toxicity. The approach promises to be a useful TIE tool for organophosphate and pyrethroid pesticides, particularly in a water matrix, and potentially could be used for identification of toxicity attributable to other pesticides.
ESTHER : Weston_2009_Environ.Sci.Technol_43_5514
PubMedSearch : Weston_2009_Environ.Sci.Technol_43_5514
PubMedID: 19708390

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 : Bridging the synaptic gap: neuroligins and neurexin I in Apis mellifera - Biswas_2008_PLoS.One_3_e3542
Author(s) : Biswas S , Russell RJ , Jackson CJ , Vidovic M , Ganeshina O , Oakeshott JG , Claudianos C
Ref : PLoS ONE , 3 :e3542 , 2008
Abstract : Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.
ESTHER : Biswas_2008_PLoS.One_3_e3542
PubMedSearch : Biswas_2008_PLoS.One_3_e3542
PubMedID: 18974885
Gene_locus related to this paper: apime-b9vmq4 , apime-b9vmq5 , apime-b9vmq6 , apime-b9vmq7 , apime-a0a088atg1