3. Results and discussion
3.3. Differences in the active-site gorge topology between insect two AChEs
differentiation in the active-site gorge between insect two AChEs during the evolution after gene duplication, the degree of changes in the amino acid residues composing the active-site gorge was estimated by measuring the levels of the
67
Table 1. LRE (Leu-Arg-Glu) motif of AChE2 in insect and their location.
* Amino acid numbering of AChE from Torpedo californica.
Order Total No.
of species LRE-motif
in AChE2 Family No. LRE-motif location*
Archaeognatha 1 1 Machilidae 1 309-311
Odonata 5 1 Libellulidae 1 528-533
Dermaptera 1 1 Forticulidae 1 256-258
Diptera 21 11 Culicidae 9 309-311
Tephritidae 2 528-533
Hemiptera 9 1 Aphididae 1 528-533
Hymenoptera 10 9
Apidae 4 309-311
Pteromalidae 3 309-311 Megachilidae 1 309-311 Formicidae 1 Close to 200
Other insects 22 - - - -
68
nucleotide diversity (Pi value) and site-specific selection (dN/dS ratio). The Pi values of the residues forming the five main domains (i.e., PAS, CAS, ABP, oxyanion hole and catalytic triad) were generally lower compared to the residues in other remaining region, indicating that these functionally crucial domains have been preserved during evolution (Fig. 5). This trend was true for both AChE1 and AChE2, and no significant difference in the Pi value levels was noticed between AChE1 and AChE2. Therefore, it appears that at least the minimum features required for the AChE functionality have been well retained in AChE2 as well as AChE1, which provides the fundamental basis for the observed AChE2 activity in most insects (Kim and Lee 2013).
The site-direct dN/dS of the residues forming the five main domains revealed that these residues of insect two AChEs have been undergone purifying selection.
These results again mean that residues forming the active-site gorge have been highly conserved in both lineages of AChE1 and AChE2 (Fig. 6). Overall degrees of dN/dS of the resisdues in AChE2 were not significantly different from those of AChE1, suggesting that no apparent amino acid sequence differentiation of active-site gorge has occurred either in AChE2 except for the aforementioned sequence differences in the PAS and ABP.
To determine whether the differences in the amino acid composition in PAS and ABP result in any differences in the active-site gorge topology, 3D structures
Fi
70
71
Figure 6. The degrees of site-direct selection posed to the amino acid residues of insect AChE1 (A) and AChE2 (B) as scored by dN/dS values. (C) Comparison of dN/dS values in the five main domains from active-site gorge between insect AChE1 and AChE2. A run on three sequences of AChEs from each five insect order (a total of 15 sequences) calculated by the MEC model. Positive selection is colored in shades of yellow, whereas purifying selection is colored in shades of magenta.
72
of insect two AChEs were generated and compared by homology modeling (Fig 7).
The resulting 3D structures with a particular emphasis on the PAS showed clear differences in the active-site gorge topology that appears to be generated by different amino acid residues in the PAS (i.e., Asp72 and Tyr121 in AChE1 vs.
Tyr121 and Met121 in AChE2). Generally, the active-site gorge entrance in insect AChE2 was narrower compared to that of AChE1 and the overall volume of gorge was smaller than that of AChE1. Similar observation was also reported in previous studies when the 3D crystal structure of D. melanogaster AChE2 was compared with that of Torpedo AChE (which is more similar to insect AChE1) (Harel et al., 2000; Wiesner et al., 2007). The other remaining difference in PAS residue composition (Ile vs. Glu) was determined to cause a noticeable alteration in the modeled structure of AChE. In addition, the structural difference caused by the different amino acid residues in the ABP (i.e., 288Cys in AChE1 vs. 288Leu in AChE2) was not visible in this model. More detailed investigation on their role in AChE structure and function remains to be studied.
Cross-comparison of amino acid residues between different hexapods and insects revealed that the Asp72 residue has been conserved in all insects and their common ancestors (i.e., hexapods), suggesting its critical role associated with the function of AChE1. In contrast, the amino acid residue at the corresponding site in AChE2 varied considerably (from Ser, Phe or Cys) and then the Tyr residue began
Figure 7. Co
Figure 7. Coontinued
74
Figure 7. Coontinued
75
Figure 7. Coontinued
76
77
to appear from Ephemenoptera (E. levis) and was fixed in insects. The finding that the functional PAS has been well preserved in AChE1 indicates that PAS is a feature more typical to AChE1 lineage and is likely associated with the AChE1-specific kinetic property. Since PAS is known to be associated with the allosteric regulation of substrate binding and the phenomenon of substrate inhibition (Toutant 1989), the observed substrate inhibition pattern in AChE1 but not in AChE2 is likely due to the differentiation of PAS between AChE1 and AChE2.
Unlike the PAS showing consistent difference in topology between AChE1 and AChE2, the backdoor that was visualized by 3D modeling was highly conserved (see orange arrow heads in Fig. 7) not only across a wide variety of hexapods and insects examined but also between AChE1 and AChE2, suggesting that this region has been conserved after duplication during evolution.
Interestingly, only exception was found in some Hymenopteran insects (Apis mellifera and Bombus terrestris), in which the formation of backdoor was not obvious in AChE1. Since the AChE backdoor is known to play an important role in cleaning the reaction products (acetate and choline), thereby enhancing the catalytic efficiency of AChE (Colletier et al., 2007; Nachon et al., 2008; Sanson et al., 2011). A detailed comparison of the deduced amino residues forming the backdoor (Trp84, Trp432, and Tyr442, Torpedo AChE numbering) or adjacent to it (Ser329 and Ile439, Torpedo AChE numbering) among a wide array of insects
78
revealed that the backdoor-forming residues of insects AChEs were highly conserved with those of Torpedo AChE (i.e., Trp84, Trp432, and Asp442) and completely conserved in all insects examined. The residues adjacent to the backdoor in insect AChE1 (Tyr329 and Met439), however, were different from those corresponding to Torpedo AChE (i.e., Ser329 and Ile439). Interestingly, the Tyr329 residue was substituted with Phe in AChE1 of A. mellifera and B.
terrestris, suggesting that this residue change likely causes the defect of backdoor in AChE1. To confirm whether this Tyr-to-Phe substitution actually alters the backdoor topology in AChE1, the reverse Phe–to-Tyr substitution was introduced into the A. mellifera AChE1 (AmAChE1) sequences and its 3D structure was predicted by homology modeling (Fig. 8). The substitution of Phe with Tyr clearly restored the backdoor topology that is similar to those of other insect AChE1. In the 3D structures of AmAChE1 with the Phe–to-Tyr substitution as well as other insect AChE1, the presence of Tyr391 (Ser329 in Torpedo AChE) appears to affect the spatial arrangement of Met503 (Ile439 in Torpedo AChE) in favor of opening the backdoor. In contrast, the substitution of Tyr391 with Phe results in the alteration of angles of Met503, blocking the backdoor formation. Therefore, the Tyr-to-Phe substitution occurred in AChE1 from some Hymenopteran insects is likely the main determining factor associated with alteration of backdoor topology.
A 3D structure modeling of AmAChE1 and AmAChE2 in a previous study also
79
80
Figure 8. Comparison of the backdoor region topology between AChE1 and AChE2 from Apis mellifera and other insects. The residues forming the backdoor or ones adjacent to it were shown in green. Blue dot circles indicated Met residue (Ile439, amino acid numbering of Torpedo AChE) that appear to affect backdoor opening. Red dot circles indicated Tyr or Phe residues (Ser329) that differ between A. mellifera AChE1 and AChE2 and likely affect the spatial arrangement of the Met residue. Backdoor topologies of AChE1 from Bombyx mori and A.
mellifera are shown in (A) and (B), respectively. Backdoor topology of A.
mellifera AChE1 with the reverse Phe-to-Tyr substitution is shown in (C). The consensus residues forming backdoor in five insect species are represented in (D).
81
reported that the side chain angle of a choline-binding site residue (Trp344 in AmAChE1 vs. Trp336 in AmAChE2) at the active-site gorge entrance was different by approximately 90°, likely resulting in the alteration of PAS conformation (Kim et al., 2012). Taken together, in addition to such alteration in the PAS topology from insect AChE1 and AChE2, the blocked backdoor topology are likely responsible for the significantly reduced catalytic efficiency of AmAChE1. Besides these AmAChE1-specific differences, the consensus differences in the PAS residues found between all insect AChE1 and AChE2 (i.e., Ile70, Asp72, and Tyr121 AChE1 vs. Glu70, Tyr72, and Met121 in AChE2) may function as additive factors associated with the significant reduction in AmAChE1 activity. Introduction of the Tyr-to-Phe substitution to other insect AChE1 (or vice versa; i.e., Phe-to-Tyr substitution to AmAChE1) singly or in combination with other substitutions at different PAS sites by in vitro mutagenesis in conjunction with functional analysis would be necessary to confirm the effects of these AmAChE1-specific substitutions on the catalytic property.
In summary, differentiation of active-site gorge, particularly at PAS and ABP, between insect AChE1 and AChE2 seems to have occurred early in the evolution, probably before the divergence of hexapods. Such differentiation in active-site gorge structure appears to be responsible for the observed differences in catalytic properties between AChE1 and AChE2. The introduction of Tyr-to-Phe
82
substitution to AChE1, causing a significant reduction of enzyme activity, appears to have occurred relatively recently and locally in some Hymenopteran insects, thereby functioning as a driving force for the functional transition from AChE1 and AChE2. Since similar cases (i.e., use of AChE2 as the main catalytic enzyme) have been reported in many other insect species, such functional transition of replacement was proposed to have been occurred with multiple origins across a wide variety of insects. A larger scale survey of mutations that are functionally similar the Tyr-to-Phe substitution in AChE1 would be required to understand the putatively multiple origins of such event.
83
APPENDIX. Information data table of aces and ace-like sequences from metazoans
Class Order Species ace1 ace2
Insecta Diptera
Aedes aegypti XM_001656927.1 XM_001655818.1
Aedes albopictus AB218421.1 AB218420.1
Anopheles gambiae Genome data BN000067.1
Anopheles darlingi Genome data
Anopheles funestus EZ976793.1 Genome data
Anopheles stephensi Genome data Genome data
Culex tritaeniorhynchus AB122152.1 AB122151.1
Culex pipiens AJ489456.1 AM159193.1
Culex pipiens pallens AY762905.1
Culex quinquefasciatus XM_001847396.1 XM_001842175.1
Phlebotomus papatasi JQ922267.1 Genome data
Musca domestica AF281162.1
Stomoxys calcitrans HM125963.1
Haematobia irritans AY466160.1
Cochliomyia hominivorax FJ830868.1
Lucilia cuprina JF776371.1
Ceratitis capitata EU130781.1
Exorista sorbillans HM028669.1
Bactrocera dorsalis AY155500.1
Drosophila melanogaster NM_057605.5
Drosophila albomicans Genome data
Drosophila ananassae Genome data
84
APPENDIX. Continued
Class Order Species ace1 ace2
Insecta
Diptera
Drosophila biarmipes Genome data
Drosophila bipectinata Genome data
Drosophila elegans Genome data
Drosophila erecta Genome data
Drosophila eugracilis Genome data
Drosophila ficusphila Genome data
Drosophila kikkawai Genome data
Drosophila miranda Genome data
Drosophila sechellia Genome data
Drosophila simulans Genome data
Drosophila yakuba Genome data
Drosophila grimshawi Genome data
Lepidoptera
Helicoverpa assulta DQ001323.1 AY817736.1
Helicoverpa armigera JF894118.1 JF894119.1
Plutella xylostella AY773014.2 AY061975.1
Chilo suppressalis EF453724.1 EF470245.1
Cnaphalocrocis medinalis FN538987.1
Bombyx mandarina EU262633.2 EF166089.1
Bombyx mori NM_001043915.1 NM_001114641.1
Melitaea cinxia GQ489250.1 GQ489251.2
Heliconius melpomene Genome data
Danaus Plexippus Genome data EHJ76382
85
APPENDIX. Continued
Class Order Species ace1 ace2
Insecta
Lepidoptera Cydia pomonella DQ267977.1 DQ267976.1
Manduca sexta Genome data Genome data
Hymenoptera
Nasonia vitripennis XM_001600408.2 XM_001605518.2
Nasonia giraulti Genome data Genome data
Nasonia longicornis Genome data Genome data
Apis mellifera XM_393751.4 NM_001040230.1
Apis florea XM_003693203.1 XM_003694182.1
Bombus impatiens XM_003488192.1 Genome data
Bombus terrestris XM_003399342.1 XM_003401864.1
Oomyzus sokolowskii HM212643.1
Megachile rotundata XM_003699292.1 XM_003701146.1
Atta cephalotes Genome data Genome data
Polyrhachis vicina JF742990.1
Acromyrmex echinatior Genome data Camponotus floridanus Genome data
Linepithema humile Genome data
Pogonomyrmex barbatus Genome data
Solenopsis invicta Genome data
Harpegnathos saltator Genome data
Siphonaptera Ctenocephalides felis FN645950.1 FN645951.1 Coleoptera Leptinotarsa decemlineata JF343436.1 JF343437.1
Tribolium castaneum HQ260968.1 HQ260969.1
86
APPENDIX. Continued
Class Order Species ace1 ace2
Insecta
Coleoptera Alphitobius diaperinus EU086057.1 EU086056.1
Lasioderma serricorne GU211888.1
Hemiptera
Aphis gossypii AB158637.1 AF502081.1
Aphis glycines JQ349160.1
Rhopalosiphum padi AY667435.1 AY707318.1
Sitobion avenae AY819704.1 AY707319.1
Myzus persicae AY147797.1 AF287291.1
Schizaphis graminum AF321574.1
Acyrthosiphon pisum XM_001948618.2 XM_001948953.2
Nilaparvata lugens HQ605041.1 JN688930.1
Nephotettix cincticeps AB264392.1 AF145235.1
Cimex lectularius JN563927.1 GU597839.1
Bemisia tabaci EF675188.1 EF675190.1
Psocoptera
Liposcelis bostrychophila FJ647185.1 EF362950.1
Liposcelis paeta GU214754.1
Liposcelis decolor FJ647186.1 FJ647187.1
Liposcelis entomophila EU854149.2 EU854150.1
Phthiraptera Pediculus humanus corporis AB266606.1 AB266605.1 Pediculus humanus capitis AB266615.1 AB266614.1 Blattodea Blattella germanica DQ288249.1 DQ288847.1 Orthoptera Locusta migratoria manilensis EU231603.1
87
APPENDIX. Continued
Class Order Species ace1 ace2
Insecta
Dermaptera Anechura japonica Partial cloning Partial cloning
Odonata
Lyriothemis pachygastra Partial cloning Partial cloning Orthetrum albistylum speciosum Partial cloning Partial cloning Sympetrum infuscatum Partial cloning Partial cloning Coenagrion lanceolatum Partial cloning Partial cloning Calopteryx atrata Partial cloning Partial cloning Calopteryx japonica Partial cloning Partial cloning Ephemeroptera Ecdyonurus levis Partial cloning Partial cloning Archaeognatha Haslundichilis viridis Partial cloning Partial cloning Zygentoma Ctenolepima longicandata Partial cloning Partial cloning
Entognatha Entomobryomorpha Orchesella villosa FJ228227.1 FJ228228.1 Tomocerus kinoshitai Partial cloning Partial cloning
Branchiopoda Cladocera Daphnia pulex Uniprot: E9FRY6 Uniprot: E9G1H3 Chilopoda Geophilomorpha Strigamia maritima Genome data Genome data
Arachnida
Ixodida
Rhipicephalus microplus AJ223965.1
Rhipicephalus appendiculatus AJ006338.1
Rhipicephalus decoloratus AJ006337.1
Dermacentor variabilis AY212906.1
Trombidiformes Tetranychus urticae GQ461353.1
Tetranychus evansi JQ779843.1
88
APPENDIX. Continued
Phylum Class Species ace1 ace2 ace3 ace4
Nematoda
Chromadorea
Caenorhabditis elegans NM_078259.6 AF025378.3 AF039650.4 NM_064561.4 Caenorhabditis brenneri uniprot:G0MGN3 uniprot: G0NVV5 uniprot:G0MVQ6 uniprot:G0MVQ7
Caenorhabditis briggsae U41846 AF030037 AF159504 AF159505.1
Caenorhabditis remanei uniprot: E3MKC8 uniprot: E3LM27 uniprot: E3LF28 uniprot: E3LF29 Heligmosomoides
GU166345.1 GU166346.1 GU166347.1
Dictyocaulus viviparus DQ375489.1 AY218789.1
Phylum Class Species ace-like 1 ace-like 2 ace-like 3 ace-like 4 Choanoflagellata Monosiga brevicollis XM_001750752.1 XM_001743828.1 XM_001749298.1
Porifera Demospongiae Amphimedon queenslandica
XM_003387193.1
Placozoa Tricoplacia Trichoplax adhaerens XM_002117107.1 XM_002117094.1 XP_002117136.1 XP_002117133.1 Cnidaria Anthozoa Nematostella vectensis XM_001629623.1 XM_001627440.1 Ctenophora Tentaculata Mnemiopsis leidyi ML073237a
Platyhelminthes
Digenea Schistosoma mansoni AF279461.1
Schistosoma haematobium AY228511.1
Schistosoma bovis AF279463.1
Trematoda Clonorchis sinensis GAA53463.1 DF144213.1 Mollusca
Gastropoda Lottia gigantea Genome data Genome data Cephalopoda Doryteuthis opalescens AF065384.1 Bivalvia Crassostrea gigas EKC37862.1
Annelida Polychaeta Capitella teleta ELU02276.1 ELU12508.1 ELU18693.1 ELT99434.1 Clitellata Helobdella robusta jgi|Helro1|76935 jgi|Helro1|120724
89
LITERATURE CITED
Ariel, N., Ordentlich A., Barak D., Bino T., Velan B., Shafferman A., 1998. The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors. Biochem J. 335: 95-102
Arnold, K., Bordoli L., Kopp J., Schwede T., 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 22:
195-201
Augustinsson, K.B., 1949. Substrate Concentration and Specificity of Choline Ester-Splitting Enzymes. Arch Biochem. 23: 111-126
Baek, J.H., Kim J.I., Lee D.W., Chung B.K., Miyata T., Lee S.H., 2005. Identification and characterization of ace1-type acetylcholine sterase likely associated with organophosphate resistance in Plutella xylostella. Pestic Biochem Phys. 81: 164-175 Ballard, C.G., Greig N.H., Guillozet-Bongaarts A.L., Enz A., Darvesh S., 2005.
Cholinesterases: roles in the brain during health and disease. Curr Alzheimer Res. 2:
307-318
Barak, D., Kronman C., Ordentlich A., Ariel N., Bromberg A., Marcus D., Lazar A., Velan B., Shafferman A., 1994. Acetylcholinesterase Peripheral Anionic Site Degeneracy Conferred by Amino-Acid Arrays Sharing a Common Core. J Biol Chem.
269: 6296-6305
Colletier, J.P., Royant A., Specht A., Sanson B., Nachon F., Masson P., Zaccai G., Sussman J.L., Goeldner M., Silman I., Bourgeois D., Weik M., 2007. Use of a 'caged' analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography. Acta Crystallogr D Biol Crystallogr. 63: 1115-1128
Eyre-Walker, A., Gaut B.S., 1997. Correlated rates of synonymous site evolution across plant genomes. Mol Biol Evol. 14: 455-460
Fournier, D., Berge J.B., Dealmeida M.L.C., Bordier C., 1988. Acetylcholinesterases from Musca-Domestica and Drosophila-Melanogaster Brain Are Linked to
90
Membranes by a Glycophospholipid Anchor Sensitive to an Endogenous Phospholipase. J Neurochem. 50: 1158-1163
Gao, J.R., Kambhampati S., Zhu K.Y., 2002. Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage. Insect Biochem Molec. 32: 765-775
Gauthier, M., Belzunces L.P., Zaoujal A., Colin M.E., Richard D., 1992. Modulatory Effect of Learning and Memory on Honey-Bee Brain Acetylcholinesterase Activity.
Comp Biochem Phys C. 103: 91-95
Gerlt, J.A., Gassman P.G., 1993. Understanding the Rates of Certain Enzyme-Catalyzed Reactions - Proton Abstraction from Carbon Acids, Acyl-Transfer Reactions, and Displacement-Reactions of Phosphodiesters. Biochemistry. 32: 11943-11952
Hall, Z.W., Sanes J.R., 1993. Synaptic structure and development: the neuromuscular junction. Cell. 72 Suppl: 99-121
Harel, M., Kryger G., Rosenberry T.L., Mallender W.D., Lewis T., Fletcher R.J., Guss J.M., Silman I., Sussman J.L., 2000. Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors.
Protein Sci. 9: 1063-1072
Harel, M., Quinn D.M., Nair H.K., Silman I., Sussman J.L., 1996. The X-ray structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase. J Am Chem Soc. 118: 2340-2346 Harel, M., Sussman J.L., Krejci E., Bon S., Chanal P., Massoulie J., Silman I., 1992.
Conversion of Acetylcholinesterase to Butyrylcholinesterase - Modeling and Mutagenesis. P Natl Acad Sci USA. 89: 10827-10831
Huchard, E., Martinez M., Alout H., Douzery E.J.P., Lutfalla G., Berthomieu A., Berticat C., Raymond M., Weill M., 2006. Acetylcholinesterase genes within the Diptera:
takeover and loss in true flies. P Roy Soc B-Biol Sci. 273: 2595-2604
Hunter, D.D., Cashman N., Morris-Valero R., Bulock J.W., Adams S.P., Sanes J.R., 1991.
An LRE (leucine-arginine-glutamate)-dependent mechanism for adhesion of neurons to S-laminin. J Neurosci. 11: 3960-3971
91
Ilg, T., Schmalz S., Werr M., Cramer J., 2010. Acetylcholinesterases of the cat flea Ctenocephalides felis: identification of two distinct genes and biochemical characterization of recombinant and in vivo enzyme activities. Insect Biochem Mol Biol. 40: 153-164
Johnson, G., Moore S.W., 2006. The peripheral anionic site of acetylcholinesterase:
Structure, functions and potential role in rational drug design. Curr Pharm Design. 12:
217-225
Johnson, G., Moore S.W., 2013. The Leu-Arg-Glu (LRE) adhesion motif in proteins of the neuromuscular junction with special reference to proteins of the carboxylesterase/cholinesterase family. Comp Biochem Physiol Part D Genomics Proteomics. 8: 231-243
Jordan, I.K., Rogozin I.B., Wolf Y.I., Koonin E.V., 2002. Essential genes are more evolutionarily conserved than are nonessential genes in bacteria. Genome Res. 12:
962-968
Kaplan, D., Ordentlich A., Barak D., Ariel N., Kronman C., Velan B., Shafferman A., 2001. Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase? Biochemistry. 40: 7433-7445
Kim, J.I., Jung C.S., Koh Y.H., Lee S.H., 2006. Molecular, biochemical and histochemical characterization of two acetylcholinesterase cDNAs from the German cockroach Blattella germanica. Insect Mol Biol. 15: 513-522
Kim, Y.H., Cha D.J., Jung J.W., Kwon H.W., Lee S.H., 2012. Molecular and Kinetic Properties of Two Acetylcholinesterases from the Western Honey Bee, Apis mellifera.
Plos One. 7
Kim, Y.H., Choi J.Y., Je Y.H., Koh Y.H., Lee S.H., 2010. Functional analysis and molecular characterization of two acetylcholinesterases from the German cockroach, Blattella germanica. Insect Mol Biol. 19: 765-776
Kim, Y.H., Lee S.H., 2013. Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta? Insect Biochem Molec. 43: 47-53
92
Kretsing, R.H., Nockolds C.E., 1973. Carp Muscle Calcium-Binding Protein: II. Structure Determination and General Description. J Biol Chem. 248: 3313-3326
Lang, G.J., Zhang X.H., Zhang M.Y., Zhang C.X., 2010. Comparison of catalytic properties and inhibition kinetics of two acetylcholinesterases from a lepidopteran insect. Pestic Biochem Phys. 98: 175-182
Lee, D.W., Kim S.S., Shin S.W., Kim W.T., Boo K.S., 2006. Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenee). Bba-Gen Subjects. 1760: 125-133
Lee, J., Jung I.D., Nam S.W., Clair T., Jeong E.M., Hong S.Y., Han J.W., Lee H.W., Stracke M.L., Lee H.Y., 2001. Enzymatic activation of autotaxin by divalent cations without EF-hand loop region involvement. Biochem Pharmacol. 62: 219-224
Li, F., Han Z.J., 2002. Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover). Arch Insect Biochem. 51: 37-45
Li, Q., Liu Y., Zhao W., Chen X.Z., 2002. The calcium-binding EF-hand in polycystin-L is not a domain for channel activation and ensuing inactivation. FEBS Lett. 516: 270-278
Lu, Y.H., Park Y., Gao X.W., Zhang X., Yao J.X., Pang Y.P., Jiang H.B., Zhu K.Y., 2012.
Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci Rep. 2
Mallender, W.D., Szegletes T., Rosenberry T.L., 2000. Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway. Biochemistry. 39: 7753-7763
Massoulíe, J., Barnard E., Chatonnet A., Bacou F., Doctor B.P., Quinn D.M., 1991.
Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology. Am Chem Soc. 394-398
Menard, R., Storer A.C., 1992. Oxyanion Hole Interactions in Serine and Cysteine Proteases. Biol Chem H-S. 373: 393-400
Mutero, A., Fournier D., 1992. Posttranslational Modifications of Drosophila Acetylcholinesterase - Invitro Mutagenesis and Expression in Xenopus Oocytes. J
93 Biol Chem. 267: 1695-1700
Nabeshima, T., Kozaki T., Tomita T., Kono Y., 2003. An amino acid substitution on the second acetylcholinesterase in the pirimicarb-resistant strains of the peach potato aphid, Myzus persicae. Biochem Bioph Res Co. 307: 15-22
Nachon, F., Stojan J., Fournier D., 2008. Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel. FEBS J. 275: 2659-2664
Olivera, S., Rodriguez-Ithurralde D., Henley J.M., 2003. Acetylcholinesterase promotes neurite elongation, synapse formation, and surface expression of AMPA receptors in
Olivera, S., Rodriguez-Ithurralde D., Henley J.M., 2003. Acetylcholinesterase promotes neurite elongation, synapse formation, and surface expression of AMPA receptors in