Insect Biochemistry and Molecular Biology 34 (2004) 353–363 www.elsevier.com/locate/ibmb
Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina R. Heidari a,b,1, A.L. Devonshire a,2, B.E. Campbell a, K.L. Bell a, S.J. Dorrian a, J.G. Oakeshott a, Dr. R.J. Russell a, a
b
CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia School of Agriculture, Charles Sturt University, PO Box 588, Wagga Wagga, NSW 2678, Australia Received 15 July 2003; received in revised form 16 January 2004; accepted 19 January 2004
Abstract Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcaE7, the gene encoding carboxylesterase E3, that enhance the enzyme’s ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme–substrate interactions and future evolution of this gene. # 2004 Elsevier Ltd. All rights reserved. Keywords: Lucilia cuprina; Esterase; Kinetics; Mutagenesis; Organophosphorus insecticides; Resistance
1. Introduction The same esterase-based resistance mechanisms to organophosphorus (OP) insecticides are found in the sheep blowfly, Lucilia cuprina, and the housefly, Musca domestica (Newcomb et al., 1997; Campbell et al., 1998a; Claudianos et al., 1999). These involve equiva Corresponding author. Tel.: +61-2-6246-4160; fax: +61-2-62464173. E-mail address:
[email protected] (Dr. R.J. Russell). 1 Present address: John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia. 2 Seconded to CSIRO Entomology from Rothamsted Research, Harpenden, Herts, UK.
0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.01.001
lent mutations in the orthologous esterase 3 (LcaE7/ E3) and aliesterase (aliE/ALI) gene/enzyme systems, respectively. In both species, modest levels of resistance (4 to 30 fold) to most OPs are due to either a Gly ! Asp mutation (G137D), or while an independent Trp251 ! Leu=Ser mutation (W251L/S), while the latter also confers higher levels of resistance (> 100-fold) to a subset of OPs, especially malathion, that have carboxylester as well as phosphoester bonds. Modelling against the known tertiary structure of the related acetylcholinesterase (AChE) molecule indicates that the G137D mutation lies in the oxyanion hole and W251L comprises part of the acyl binding site within the active site of the insect esterases (Newcomb et al.,
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1997; Campbell et al., 1998a). AChE and both insect esterases are members of the carboxyl/cholinesterase multigene family, which is characterised by an a/b hydrolase fold tertiary structure and a reaction mechanism involving a charge relay system mediated by a Ser-His-Glu/Asp catalytic triad (Ollis et al., 1992; Oakeshott et al., 1995, 1999). The oxyanion hole comprises three small residues, generally two glycines and an alanine, whose peptide >NH groups hydrogen bond to the carbonyl oxygen of carboxylester substrates. The acyl binding site is a hydrophobic region that accommodates at least part of the acid component of the ester when it binds to the catalytic serine (Ja¨rv, 1984; Sussman et al., 1991). Hydrolysis of carboxylester substrates proceeds via a two-step reaction mechanism (Ja¨rv, 1984) involving covalent binding of the carbonyl carbon of the substrate to the catalytic serine with loss of the alcohol side chain to generate an acyl-enzyme intermediate, followed by nucleophilic attack by a water molecule on the carbonyl-serine bond to liberate the acid and regenerate free enzyme. These esterases are generally inhibited irreversibly by OPs because the second step occurs very slowly or not at all. This appears to depend on the different orientation needed for the bound water molecule to attack the phosphorylated intermediate, which has a tetrahedral structure around the phosphorus atom in contrast to the planar carbonyl in an acyl intermediate (Ja¨rv, 1984). Newcomb et al. (1997) and Campbell et al. (1998a) proposed that the G137D mutation in the oxyanion hole alters the orientation of this bound water molecule, facilitating attack on a phosphorylated serine but diminishing that on an acylated serine, consistent with the marked loss of carboxylesterase activity in the mutant. Campbell et al. (1998a) and Devonshire et al. (2003) further proposed that the W251L mutation in the acyl binding pocket creates more space to accommodate substrates with bulky acid groups and reduces the steric hindrance to the inversion that must occur around the phosphorus during hydrolysis of OPs. The additional space conferred by this mutation should also allow hydrolysis of bulky carboxylester substrates like malathion even though ‘oriented’ differently from typical carboxylesterase substrates in having a much larger acid than alcohol component to the ester. Devonshire et al. (2003) determined the hydrolysis kinetics of O,O-diethyl and O,O-dimethyl OPs, which represent the overwhelming majority of this class of insecticides, by the wild type and natural resistance mutants of E3 using fluorogenic substrates with a coumarin leaving group. First-order rate constants (kcat) for the G137D enzyme were only approx. 0.05 min1 both for the diethyl (dECP) and dimethyl (dMUP) analogs. Nevertheless, these rates were 30–50 times higher than for the wild type enzyme and calculations
showed that this can provide sufficient detoxification capability in vivo to account for the modest levels of resistance observed. Values for the W251L enzyme were 0.009 min1 for dECP and 0.06 min1 for dMUP. This mutant enzyme also retained good malathion carboxylesterase (MCE) activity with a Km of 1.09lM and a kcat of 220 min1 compared to 0.33 lM and 54 min1 for wild type E3, so that the specificity constant was only slightly changed. The high level of malathion resistance conferred by this enzyme was explained by the combined effect of the marginally improved MCE activity and the acquired OP hydrolase activity against malaoxon, the in vivo activated form of the insecticide. This paper explores the potential for generating more effective OP hydrolysing forms of E3 by making 14 other mutations in and around the oxyanion hole and acyl binding pocket and analysing their dECP, dMUP and MCE hydrolytic activities. Our choice of mutations was influenced by structural and mutational studies of the well-characterised AChE and butyrylcholinesterase (BuChE) enzymes (Taylor and Radic, 1994, and see also the ESTHER website (http://bioweb.ensam.inra.fr/ ESTHER/general?what=index) and comparative sequence analyses of the carboxyl/cholinesterase multigene family (Oakeshott et al., 1999; Claudianos et al., 2001). Of particular relevance are a series of studies designing human BuChE mutants with enhanced ability to hydrolyse OPs (Millard et al., 1995; Lockridge et al., 1997; Millard et al., 1998).
2. Materials and methods Methods for making, expressing and assaying mutants, described fully by Devonshire et al. (2003), are outlined here. 2.1. Mutagenesis and expression Mutations were introduced into the cloned, OP susceptible E3 gene (Newcomb et al., 1997) by sitedirected mutagenesis with the QuickChangeTM kit using the manufacturer’s (Stratagene) protocols. Details of primers are available on request. All mutant genes were fully sequenced by standard dye terminator technology to ensure that the desired mutations had been introduced with no unintended mutations. Validated mutants were then transferred into the baculovirus vector pFastBac1 (GibcoBRL Life Technologies) and these constructs plus the vector-only and b-glucuronidase (GUS) controls were expressed after optimisation in Sf9 cells using HyQSFX insect serum free medium (HyClone). Cells were lysed and stored as v 25 ll aliquots at 80 C. Two to five replicate extracts were analysed for each substrate to determine kinetic constants (see below). Most replicate extracts were
R. Heidari et al. / Insect Biochemistry and Molecular Biology 34 (2004) 353–363
prepared from the same cultures but some from replicate cultures were also assayed. Variation between replicate cultures was not significantly (95% confidence limits) greater than that between replicate extracts of the same culture. 2.2. Naphthyl acetate hydrolysis Hydrolysis of 1-naphthyl acetate was assayed spectrophotometrically in microplates using both endpoint (Devonshire et al., 2003) and continuous assays (Grant et al., 1989) at a range of substrate concentrations spanning the Km (based on preliminary assays), and the data fitted to a nonlinear regression to determine kinetic constants (Enzfitter 1.05 software, Elsevier-Biosoft). The molar amount of esterase in assays was determined by titration with a fluorogenic inhibitor (see below) and used to calculate kcat values. 2.3. Esterase titration and OP hydrolase activity The hydrolysis of diethyl- and dimethyl-phosphates by the cell extracts was determined fluorometrically during incubation with O,O-diethyl O-(2-oxo-2H-chromen-7-yl) phosphate (dECP) and O,O-dimethyl 4methylumbelliferyl phosphate (dMUP). The rate of release of the coumarin ‘leaving group’, expressed as fraction of the initial ‘burst release’ of fluorescence as the esterase rapidly becomes phosphorylated, gives a direct measure of the kcat values (Devonshire et al., 2003), which describe the maximal rate of hydrolysis of any OP having dimethyl or diethyl substituents. The assay requires high concentrations (0.1 mM) of these compounds to ensure the enzyme is saturated, as judged by the rate of fluorescence increase becoming linear (Devonshire et al., 2003). Whilst the analysis of
Fig. 1.
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reaction rates at lower dECP/dMUP concentrations could enable measurement of Km values, these would be of relevance only to these particular model compounds. The data from these assays also provided a measure of the molar amount of esterase (from the ‘burst release’ of fluorescence) for use in calculating kcat values for 1-naphthyl acetate and malathion. Only those extracts with esterase titres at least twice that of the vector-only and GUS controls (0:4 0:1 pmol=ll) were analysed further. Any contribution from the very low hydrolase activity in controls (Devonshire et al., 2003) was thus minimised in the assays. 2.4. Malathion carboxylesterase (MCE) activity MCE activity was assayed as described by Campbell et al. (1998a) and Devonshire et al. (2003) without diluting the specific activity of the 14C malathion (25 mCi mmol1) for enzymes that appeared to have a low Km, and the data again fitted to a nonlinear regression to determine kinetic constants, incorporating esterase titration data to enable calculation of kcat values. 2.5. Substrates used The structures of the substrates used are given in Fig. 1.
3. Results and discussion We have examined mutations at seven amino acid residues in regions of E3 corresponding to three distinct subsites of the known AChE active site; the oxyanion hole (E3 residue G137), the anionic site (E3 residues Y148, E217 and F354) and acyl binding pocket (E3 residues P250, W251 and F309). The anionic site and
Molecular structures of substrates used.
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acyl binding pocket correspond to the p1 and p2 subsites, respectively, in the nomenclature of Ja¨rv (1984). Table 1 gives the correspondence between these and other key active site residues in E3 and the three thoroughly characterised Torpedo californica, D. melanogaster and human AChE structures. Tables 2 and 3 show the effects of the various mutations in E3 on Km and kcat for 1-naphthyl acetate esterase and MCE activities, respectively. Table 4 shows their effect on OP hydrolase activity in terms of kcat values for dECP and dMUP. Data for expressed enzymes corresponding to the wild type (WT) and natural mutant enzymes have been presented earlier (Devonshire et al., 2003) but are included here for completeness. 3.1. Mutations in the oxyanion hole In TcAChE, the oxyanion hole comprises G118, G119 and A201, which corresponds to G136, G137 and A219 in E3. These residues are highly conserved throughout the carboxyl/cholinesterase multigene family (Oakeshott et al., 1999) and there is empirical evidence for the conservation of the oxyanion hole structure from X-ray crystallographic studies of several cholinesterases and lipases Cygler and Schrag, 1997, albeit the structure does change during interfacial activation in some lipases (Derewenda et al., 1992). There is also empirical structural evidence for their function Table 1 Catalytically important residuesa conserved in esterases (LcE3 residues mutated in this study are shown in bold and conservative differences from TcAChE are in italics). E3 and its ortholog in D. melanogaster (EST23) share the same residue numbering Esterase (% identity to TcAChE)
LcE3 (29%)
TcAChE
DmAChE (36%)
Human AChE (54%)
Catalytic triad
S218 E351 H471
S200 E327 H440
S238 E367 H480
S203 E334 H447
Oxyanion hole
G136 G137 A219
G118 G119 A201
G150 G151 A239
G121 G122 A204
p1 subsite (‘anionic site’)
Y148 E217 F354 F355
Y130 E199 F330 F331
Y162 E237 Y370 F371
Y133 E202 Y337 F338
p2 subsite (acyl binding pocket)
P250 W251 V307 F309 F421
P232 W233 F288 F290 V400
P270 W271 L328 F330 F440
P239 W240 F295 F297 V407
a All numbering is based on Newcomb et al. (1997) for LcE3, Sussman et al. (1991) for TcAChE, Harel et al. (2000) for DmAChE and Ordenlitch et al. (1993) human AChE.
Table 2 Hydrolysis kineticsa of 1-naphthyl acetate by LcE3WT and its mutants expressed in vitro Enzymes
Km (lM)
kcat (sec1)
kcat/Km (lM1sec1)
73 2 E3WT Oxyanion hole mutants 22 4 E3G137D 92 14 E3G137E 114 17 E3G137Hb 78 9 E3G137R
248 7
3:4 0:2
25 2 114 7 55 4 166 10
1:14 0:2 1:24 0:4 0:48 0:03 2:13 0:3
p1 subsite (‘anionic site’) 24 3 E3Y148Fb 19 1 E3E217M 29 6 E3F354W 27 3 E3F354L
129 17 10 1 514 25 30 3
5:38 1:4 0:53 0:04 17:7 2:6 1:1 0:1
p2 subsite (acyl pocket) 133 17 E3W251L 180 1 E3W251S 464 25 E3W251T 90 10 E3W251G 251 4 E3W251A 22 4 E3F309L
149 20 249 42 248 18 264 44 503 29 297 40
1:12 0:1 1:38 0:2 0:53 0:02 2:93 0:6 2:0 0:1 13:5 4:5
Double mutants E3Y148F/G137D E3P250S/W251L E3W251L/G137D E3F309L/W251Lb
23 2 57 9 40 5 112 2
0:77 0:04 0:73 0:2 0:18 0:03 0:75 0:03
30 4 78 30 217 26 150 4
a All values are means (SE) for 3–5 independent determinations, except where indicated. b Values are means of replicate experiments.
in stabilising the oxyanion formed by the carbonyl oxygen of the carboxylester substrate in the first transition state during catalysis (Grochulski et al., 1993; Martinez et al., 1994). This stabilisation is achieved by a network of hydrogen bonds to the amide >NH groups of these three residues in the peptide chain (Ordentlich et al., 1998). Koellner et al. (2000) have shown that the peptide chain amide groups of the Gly residues in the AChE oxyanion hole also make hydrogen bonds with buried structural water molecules that are retained during catalysis and thought to act as lubricants to facilitate traffic of substrates and products within the active site. We have made three mutations to the G137 of E3 in addition to G137D found naturally in OP resistant L. cuprina; G137E as the other acidic amino acid, G137H because His is also mainly non-protonated at neutral pH (pKa 6.5, compared to 4.4 for Asp and Glu, which will be negatively charged) and it was found to confer OP hydrolase activity on human BuChE when substituted for either Gly in its oxyanion hole (Lockridge et al., 1997), and G137R to examine the effects of the most strongly basic substitution possible (Arg has pKa around 12).
R. Heidari et al. / Insect Biochemistry and Molecular Biology 34 (2004) 353–363 Table 3 MCE kineticsa for LcE3WT and its mutants expressed in vitro Enzyme
Km (lM)
kcat (min1)
0:33 0:08 E3WT Oxyanion hole mutants 13:8 4:4 E3G137Db 8:7 1:6 E3G137E 34:2 4:1 E3G137Hc 9:6 2:9 E3G137R
7:8 0:6 7:2 0:6 11:4 0:6 678 234
0:57 0:2 0:82 0:1 0:33 0:02 70:6 8
p1 subsite (‘anionic site’) 1:11 0:04 E3Y148F 0:65 0:13 E3E217M 0:64 0:08 E3F354W 3:1 0:57 E3F354L
199 19 3:0 0:18 68 6 20 3
p2 subsite (acyl pocket) 1:09 0:4 E3W251Lb 0:24 0:02 E3W251Sc 0:13 0:03 E3W251T 0:09 0:01 E3W251G 0:21 0:02 E3W251A 2:2 0:12 E3F309Lc Double mutants E3W251L/G137Db 1:5 0:2 1:1 0:6 E3W251L/F309L 3:3 0:1 E3Y148F/G137D 0:65 0:13 E3P250S/W251L
b
54 6
kcat/Km (lM1 min1)
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Table 4 Titre and kcata for hydrolysis of OPs by LcE3WT and its mutants expressed in vitro Enzyme
164 21
Titre (pmol ll1)
dECP kcat (min1)
dMUP kcat (min1)
E3WTb 5:9 0:26 Oxyanion hole mutants 4:2 0:13 E3G137Db E3G137E 6:5 0:22 E3G137H 2:5 0:06 E3G137R 1:1 0:01
0:0009 0:0001
0:0018 0:0002
0:0500 0:0049 0:0045 0:0005 0:0120 0:0006 nac
0:0570 0:0003 0:0038 0:0011 0:0054 0:0007 nac
179 11 4:62 0:5 106 12 6:39 0:3
p1 subsite (‘anionic site’) 7:6 2:5 E3Y148Fd E3E217M 5:6 0:64 E3F354W 7:5 2:19 E3F354L 1:7 0:1
0:0005 0:0002 0:0014 0:0000 0:0005 0:0001 0:0057 0:0001
0:0020 0:0003 0:0022 0:0000 0:0032 0:0000 0:0045 0:0005
220 17 93 25 34:8 1:8 307 31 68:4 7:2 172 44
202 21 387 42 267 34 3406 380 325 39 78 6
p2 subsite (acyl pocket) E3W251Lb 6:9 2:0 E3W251Sd 6:2 0:28 E3W251T 1:6 0:12 E3W251G 5:7 0:21 E3W251A 8:2 0:39 E3F309L 1:1 0:07
0:0092 0:0004 0:0065 0:0004 0:0115 0:0006 0:0030 0:0005 0:0185 0:0004 0:0047 0:0009
0:0610 0:0060 0:0315 0:0025 0:1822 0:0205 0:0046 0:0008 0:0807 0:0084 0:0034 0:0009
5:4 1:2 147 8:4 2:4 0:24 53:4 2:4
3:6 0:9 133 15 0:73 0:1 82 9
Double mutants E3W251L/ G137Db E3W251L/F309L E3Y148F/G137D E3P250S/W251L
0:0210 0:0013 0:0049 0:0002 0:0268 0:0015 0:0036 0:0003
0:0530 0:0025 0:0159 0:0006 0:0439 0:0002 0:0180 0:0000
a All values are means (SE) for 3–5 independent determinations, except where indicated. b Data are taken from Devonshire et al. (2003). c Values are means of replicate experiments.
G137D was previously shown to diminish the carboxylesterase activity of E3 against both 1-naphthyl acetate and malathion (Newcomb et al., 1997; Campbell et al., 1998a; Devonshire et al., 2003), more so for malathion, particularly through effects on Km, although kcat was also impaired (see also Tables 2 and 3). Devonshire et al. (2003) suggested that the large acyl group of malathion (ethyl succinate dimethyl phosphate) is unlikely to fit comfortably in the acyl binding pocket of most characterised carboxyl/cholinesterases and might therefore require a quite different orientation, as proposed for juvenile hormone in JH esterase (Thomas et al., 1999). In so doing, its hydrolysis could be severely compromised not only by reorientation of a bound water, but also by substitution of Gly by the bulky acidic Asp in the oxyanion hole. We now find that G137E and G137H behave very similarly to G137D with 1-naphthyl acetate and malathion as substrates, again consistent with an adverse effect on transition state formation and compounding difficulties in accommodating the bulky acyl group of malathion. The G137R mutant had relatively little effect on Km and kcat for 1-naphthyl acetate hydrolysis, but larger
2:5 1:1 7:4 0:76 6:6 0:75 6:7 0:80
a all values are means (SE) for 3–5 independent determinations, except where indicated. b data are taken from Devonshire et al. (2003). c No activity detectable. d Values are means of replicate experiments.
and opposing effects on the two components of the kinetics for MCE activity. Km was higher, as for the other mutants, but in this case kcat was also substantially higher, leaving the specificity constant (kcat/Km) only about twofold less than for the wild type enzyme. This mutant had the best kcat for malathion of any of those studied. We cannot fully explain these results but note that Arg often complexes with water, acting as a general acid base. Also its relatively large side chain is long and flexible, so that it might be able to fold out in such a way as to avoid the potential disruption to catalysis by its protonated terminal >NH2+ group or indeed hold the malathion, once bound, in an orientation favouring nucleophilic attack by water. Devonshire et al. (2003) reported greater than 30fold increases in kcat for both OP compounds, dECP and dMUP, for G137D compared to wild type E3. In kinetic terms, however it is still very slow for both substrates (around three turnovers per hour), albeit sufficient to confer significant OP resistance in vivo. Newcomb et al. (1997) suggested that the OP activity was acquired because the negatively charged Asp can
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hold and activate a water molecule in an appropriate orientation for nucleophilic attack on the phosphorus atom in the phosphorylated enzyme intermediate. A similar explanation was invoked to explain the OP hydrolase activity of the G ! D and G ! H mutations in the oxyanion hole of BuChE (Lockridge et al., 1997). The recent finding of five water molecules hydrogen-bonded with the three contiguous Gly residues (G117–119) in the crystal structure of Torpedo AChE (Koellner et al., 2000) underlines the importance of their role in catalysis. None of the other three oxyanion hole mutations enhanced OP hydrolase activity as much as G137D. Consistent with the models for G137D above, substitution by the basic Arg abolished all detectable activity. G137E and G137H showed only slightly elevated activities compared to wild type, perhaps because in E3 the additional bulk of their side chains compromised the required orientation of the water molecule achieved with the Asp substitution. In BuChE, the corresponding Glu mutation was again relatively inactive but His substitution enhanced OP hydrolase activity more so than the Asp mutant (Lockridge et al., 1997, C.A. Broomfield, pers. comm.), reflecting a likely oxyanion hole conformation distinct from that of LcE3. It is remarkable that mutations in AChE from culicine and anopheline mosquitoes (Weill et al., 2003) at sites corresponding to TcAChE Gly119 and LcE3 Gly137 have very recently been associated with target site insensitivity to carbamate and OP insecticides. Intriguingly, the mosquito mutation changes the Gly to a Ser residue in resistant individuals, although the same change segregates among susceptible individuals in AChE in the mite Tetranychus urticae (Anazawa et al., 2003). Whilst Weill et al. (2003) do not propose that the mutation confers OP hydrolytic activity, their results would at least imply some change to the access or binding of the compounds to the catalytic Ser. Clearly, more work will be needed to understand the modes of action of this mutation in AChE but it would also now be interesting to test for effects of a Gly137Ser change on the OP hydrolase and MCE activities of carboxylesterases like E3. 3.2. Mutations in the acyl binding pocket (p2 subsite) The acyl binding pockets, or p2 subsites in Ja¨rv’s (1984) nomenclature, of structurally characterised cholinesterases are formed principally from four non-polar residues, three of which are generally also aromatic (Table 1). Together they create a strongly hydrophobic pocket to accommodate the acyl moiety of bound substrate (see above). These four residues in TcAChE, W233, F288, F290 and V400, correspond to W251, V307, F309, and F421 in E3. Similar arrays of hydrophobic residues appear to be conserved at the corre-
sponding sites of most carboxyl/cholinesterases, albeit the regions corresponding to TcAChE 288–290 can be difficult to align unambiguously (Oakeshott et al., 1993; Robin et al., 1996; Harel et al., 2000). In particular, Trp is strongly conserved at residue 233, and 290 is Phe in AChEs and most carboxylesterases, albeit a Leu or Ile in several lipases and a few carboxylesterases. BuChEs have branched chain aliphatic amino acids at positions corresponding to TcAChE 288 and 290 (Gentry and Doctor, 1991). The acyl binding pocket is substantially devoid of buried water molecules (Koellner et al., 2000). Mutational studies in several cholinesterases of the residues corresponding to TcAChE F288 and F290 confirm their key role in determining aspects of substrate specificities related to acyl group identity. In human AChE, replacement of the Phe at either position with a smaller residue like Ala improves the kinetics of the enzyme for propionyl- and butyryl-(thio)choline, which have larger acyl groups than the natural substrate (Ordentlich et al., 1993). In AChE from D. melanogaster and the housefly M. domestica, natural mutations of their F290-equivalent to the bulkier polar Tyr contribute to target site OP resistance through a marginally lower sensitivity to OPs together with a greater affinity for acetylthiocholine (Fournier et al., 1992; Walsh et al., 2001). For D. melanogaster AChE, substitution of this Phe by Leu or Ile gave an increase in OP sensitivity, although surprisingly replacement with other small aliphatic residues like Gly, Ser or Val gave, if anything, a slight decrease (Villatte et al., 2000). W233 has received much less attention in mutational studies of cholinesterases but our prior work on E3 shows its replacement with the smaller Leu residue found naturally in OP resistant strains again increases reactivity for carboxylester substrates with bulky acyl moieties as in malathion, or for OPs (Campbell et al., 1998a,b; Devonshire et al., 2003). A mutation to Gly has also been found in a homolog from the wasp Anisopteromalus calandrae that shows enhanced MCE kinetics (Zhu et al., 1999), while a Ser has been found in a homolog from M. domestica that may be associated with malathion resistance (Claudianos et al., 2001; Scott and Zhang, 2003). In respect of OP hydrolase activity, Devonshire et al. (2003) proposed that the particular benefit of such mutations is to accommodate the inversion about the phosphorus that must occur during hydrolysis of the acylated enzyme. Notably they found that the kcat for OP hydrolase activity of E3W251L is an order of magnitude higher for dMUP, a dimethylphosphate, than for dECP, a diethylphosphate. This suggests that there remain tight steric constraints on the inversion even in a mutant with a larger acyl pocket. We have mutated both the W251 and F309 residues of E3 as well as the P250 immediately adjacent to W251. In addition to the previously characterised natural W251L mutation, we have now analysed substitutions
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with four other small amino acids in W251S, W251G, W251T and W251A. A double mutant of W251L and P250S was also analysed, because this combination is found in a natural variant of the ortholog of E3 in M. domestica with high MCE (V. Taskin, RJR and JGO unpublished data). Only one F309 substitution was examined, F309L. This residue, and F307 (equivalent to F288 and F290 in TcAChE) are considered to delimit the ‘size’ of the acyl binding pocket, and replacement of either (or both, as in various vertebrate BuChEs) with aliphatic amino acids is known to relax the spatial constraints on substrate size (Gentry and Doctor, 1991; Ordentlich et al., 1993; Harel et al., 2000). Also the residue corresponding to F290 is aliphatic in some lipases (Schrag et al., 1991; Gjellesvik et al., 1994). These considerations led us to explore whether the F309L mutation would enhance MCE and OP hydrolyse activities. F309L was analysed alone and as a double mutant with W251L. The W251L mutation has had little effect on 1-naphthyl acetate hydrolysis (Devonshire et al., 2003 and Table 2), which is unsurprising since the acyl pocket only needs to accommodate an acetyl group for this substrate. Consistent with this, none of the other four mutations to small amino acids at this position has a major effect on 1-naphthyl acetate kinetics. None was as efficient as the wild type enzyme although the differences were generally small. There were no obvious differences between the two polar (S, T) and three nonpolar substitutions (L, G and A). In line with the W251L results, all four of the other 251 substitutions show some increase in specificity constant for MCE compared to wild type. Notably, all involve smaller residues than Leu and yield specificity constants higher than that for W251L. Even then, the increase is small for all but W251G; substituting this smallest amino acid gives a very marked (20-fold) increase in specificity constant over wild type enzyme. This is in good accordance with the presence of this natural mutation in some strains of A. calandrae that have strong and specific malathion resistance (Zhu et al., 1999), providing good evidence for its functional significance in these field populations of hymenopterous parasitoid. Unlike the W251L mutation in L. cuprina and M. domestica, which confers resistance by enhancing both OP hydrolase and MCE activities (Devonshire et al., 2003), the Gly mutation has only a marginal effect on dimethyl-OP hydrolase activity (Table 4). If this mutation affects the A. calandrae esterase in the same way, malathion resistance in this species would depend primarily on a dramatically improved MCE activity (Table 3). The kinetics of the W251 series of mutants with dECP and dMUP are strongly, albeit not perfectly, correlated with each other and poorly correlated with their MCE kinetics. Values of kcat with dECP and
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dMUP for W251G are substantially less than for W251L and only about threefold higher than for wild type E3. W251S also yields values lower than W251L, although markedly better than W251G for dMUP. On the other hand W251T and W251A give values clearly higher than W251L. W251T gives the highest value for dMUP, threefold better than W251L and 100-fold that of the wild type enzyme. W251A is highest for dECP, twice as good as W251L and 200-fold that of the wild type. One clear conclusion from the 251 single mutant series is that substituting a smaller amino acid than Trp enhances both MCE and dECP/dMUP kinetics. Clearly however, the steric requirements for effective acyl group accommodation reflected in the MCE results are not the same for this planar ester as those for effective inversion around the tetrahedral phosphorus reflected in the dECP/dMUP results. Compared to W251L alone, the combination of W251L with the P250S substitution slightly reduces 1-naphthyl acetate and MCE activities and gives threefold lower activities for dECP and dMUP. This could be because of a change in the conformation of the peptide chain brought about by removal of the turninducing Pro residue. These findings suggest that the P250S substitution in the high MCE variant of the M. domestica ortholog of E3 is not associated with its high MCE activity. Indeed, the corresponding residue in other insect esterases is quite variable, even though Pro is common in this position immediately upstream of the Trp in the acyl pocket (Claudianos, 1999). The specificity constant for 1-naphthyl acetate for the single F309L mutant is three times that of the wild type enzyme but in the F309L/W251L double mutant it is less than a quarter of that for W251L alone. Thus the two F309L enzymes differ by 20-fold, with both Km and kcat contributing to the effect. This implies an interaction between the residues at 251 and 309, which might be expected by analogy with the proximity of corresponding residues in the TcAChE acyl binding pocket. The apparently adverse effect of double substitution of the smaller Leu residues at both sites may be because there is then a less tight interaction between the acetyl group of 1-naphthyl acetate and the hydrophobic residues of the acyl pocket. Also, studies of housefly AChE have shown that mutating the corresponding F327 (MdAChE numbering) to tyrosine, as occurs naturally in OP resistant flies, has little effect on OP or carbamate kinetics, but gives a marked improvement in substrate kinetics (Walsh et al., 2001). Similar studies with vertebrate AChE (discussed in that paper) show that mutation of the residues corresponding to 307 and 309 in E3 has a marked effect on substrate affinity. F309L has little effect on MCE kinetics, either as a single mutant compared to wild type or as a double
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mutant with W251L compared to W251L alone. If anything, there is a negative effect, against the expectations that the greater space generated in the acyl pocket would favour accommodation of the bulky ethyl succinate dimethyl phosphate group of malathion. Compared with wild type, F309L as a single mutant shows elevated kcat values for dECP and, to a lesser extent, dMUP. However, compared to W251L, the double mutant shows very clearly poorer values for both substrates. As with the carboxylesterase activities, the OP hydrolase results show a clear interaction between the F309 and W251 substitutions. Overall, we have much less evidence for 309 than for 251 that substituting a smaller Leu residue enhances the activity of the presumptive acyl pocket of E3 to accommodate bulky acyl groups or to facilitate inversion around the phosphorus during hydrolysis of the phosphorylated enzyme. As with 251, a more extensive series of even smaller substitutions might be needed to show any marked effect. 3.3. Mutations in the p1 subsite (corresponding to the AChE anionic site) The anionic site of cholinesterases, or the p1 subsite in the original nomenclature of Ja¨rv (1984), principally comprises W84, E199 and F330, with F331 and Y130 (TcAChE nomenclature) also involved; the p electrons in these aromatic sidechains serving to accommodate the quaternary nitrogen of acetylcholine. Except for E199, immediately adjacent to the catalytic Ser 200, it is thus a highly hydrophobic site. The key residues are highly conserved across cholinesterases and, notwithstanding the likely non-ionic nature of many of their different substrates, most carboxylesterases (Oakeshott et al., 1993; Ordentlich et al., 1995; Robin et al., 1996; Claudianos et al., 2001). Except for W84 (carboxylesterases lack an 8- to 11-amino acid region encompassing TcAChE W84), E3 has identical residues to TcAChE at the corresponding LcE3 positions E217, F354 and Y148 (Table 1). Interestingly, the residue equivalent to TcAChE F330 is Leu in some lipases and certain carboxylesterases whose substrates have small alcohol leaving groups (Thomas et al., 1999; Campbell et al., 2001; Claudianos et al., 2001). Structural and mutational studies have shown that the anionic site of cholinesterases functions mainly in the first, enzyme acylation stage of the reaction (Shafferman et al., 1992; Ordentlich et al., 1995, 1996; Koellner et al., 2000; Harel et al., 2000; Brochier et al., 2001), particularly in the formation of the non-covalent transition state (Nair et al., 1994). Therefore, mutations of the key residues affect mainly Km rather than kcat. Residues in this region, especially TcAChE F330, also contribute to the binding of the anti-Alzheimer
drugs, Aricept and huperzine, and other aromatic ligands (Greenblatt et al., 2000). Studies with OP inhibitors suggest that the anionic site of cholinesterases also accommodates one of the alkoxy side chains but there is some evidence that at least part of the site may also then affect the reactivity of the phosphorylated enzyme (Qian and Kovach, 1993, and see also Ordentlich et al., 1996). There has been little mutational analysis of the corresponding p1 subsites (this nomenclature is preferable to ‘anionic site’ in relation to non-cholinesterases) among carboxylesterases but one interesting exception involves the EST6 carboxylesterase of D. melanogaster which has a His at the equivalent of Glu 199. A synthetic mutant in which this His is replaced by Glu shows reduced activity against various carboxylester substrates but has acquired some acetylthiocholine hydrolytic activity (Myers et al., 1993). The E4 carboxylesterase of the aphid Myzus persicae has a Met at this position but it is not known whether this contributes to this enzyme’s very high affinity for OPs (Devonshire, 1977). Likewise, juvenile hormone esterase has Gln instead of Glu at this position (Thomas et al., 1999). Similarly, a Y148F (TcAChEY130) substitution is one of several recorded in the E3 ortholog in an OP resistant strain of M. domestica but it is not known whether this change directly contributes to OP hydrolase activity (Claudianos et al., 1999). We have mutated the Y148, E217 and F354 residues in E3. E217M and Y148F mutations were made to explore whether the corresponding mutations in the M. persicae and M. domestica enzymes above might contribute directly to their OP reactivity. Y148F was also tested in a G137D double mutant since this is the combination found in the resistant M. domestica. F354 was mutated both to a smaller Leu residue and a larger Trp, Leu commonly being found at this position in lipases (see above). The behaviors of F354W and F354L with the two carboxylester substrates do not fit consistently well with expectations based on the AChE anionic site. One of the substitutions adds a larger aromatic side chain while the other inserts a small aliphatic side chain. One of the substrates (1-naphthyl acetate) has a bulky aromatic leaving group while the other (malathion) has only a small alkyl leaving group. The AChE model would therefore predict some effect of the two mutants on Km, but only small differences are seen for the two substrates. In fact, there are changes in Km for both mutants and substrates but they are similar in directions for the two mutants and except for a 10-fold increase for F354L with malathion, only two- to threefold in size. Conversely, the AChE model would not predict effects on kcat but there are changes of similar scale in this kinetic constant, the biggest being a 10fold decrease for F354L with 1-naphthyl acetate. These
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data suggest a different function for F354 in the active site of E3 from that of its equivalent F330 in TcAChE. Some of these differences may be because the equivalent of the W84 region in AChE is missing in E3 and F354 exerts much of its function in the AChE anionic site through its interaction with W84 (Shafferman et al., 1992; Ordentlich et al., 1995). Simple extrapolation from the AChE model would also suggest that substitutions in the p1 site of E3 would affect Km values for OP substrates, which our assay did not measure, but have little effect on kcat values, which we have measured. However, Ja¨rv (1984) suggested that the leaving groups of OPs might need at least in part to be accommodated in different areas than those of carboxylesters. Furthermore, molecular modelling of soman bound to the active site serine of TcAChE (Qian and Kovach, 1993) pointed to a strong van der Waals interaction between TcAChE F330 and the t-butyl group of the OP. In fact, we do find evidence for some interaction between LcE3 F354 and the alkyl groups of dECP/dMUP; for both substrates there are effects of 354 substitutions on kcat. Except for F354W with dECP where there is no difference, kcat is two- to sixfold higher than for wild type enzyme. Notably the substitution with the smaller Leu residue gives the greater increase, which is consistent with the pattern seen for some of the acyl binding site substitutions. It would clearly now be interesting to examine smaller residues at 354, and their combinations with acyl pocket substitutions at W251, F309 and possibly also the G137 in the oxyanion hole. The final two amino acids mutated, E217 (TcAChE E199) and Y148 (TcAChE Y130), interact directly with each other in AChE, where Tyr undergoes aromatic interaction to orientate TcAChE W84 and also hydrogen bonds through water to the Glu to stabilise its conformation in the anionic subsite; the correct orientation of the negative charge on this Glu is considered important in acylation, phosphorylation, carbamoylation, ‘ageing’ and for interaction with some non-covalent ligands (Ordentlich et al., 1995). Whilst this Glu immediately ‘upstream’ of the catalytic serine is conserved in cholinesterases, it shows some degree of variability in carboxylesterases from insect species (Field et al., 1993; Thomas et al., 1999), suggesting it plays a less critical role in this broader subclass of hydrolases. The LcE3 E217M mutation, introduced because Met is found at this position in esterase E4 of susceptible and resistant aphids M. persicae (Field et al., 1993), substantially reduces the specificity constants for both 1-naphthyl acetate and MCE activities, mainly due to effects on kcat. As with F354, there is little evidence that this residue in E3 affects acylation and the dynamics of the leaving group in the same way as does its equivalent in the cholinesterases. E217M has no detectable effect on dECP and dMUP hydrolysis, so
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any effect the equivalent methionine might have in the M. persicae E4 enzyme is not manifest in E3. In contrast to the marked adverse impact of the corresponding mutation on AChE interaction with substrates and inhibitors (Ordentlich et al., 1995), Y148F has no overall effect on the specificity constants for either 1-naphthyl acetate or malathion, or on dECP or dMUP hydrolysis, as either a single mutant or a double mutant with G137D. It is perhaps surprising that no clearer effect emerged for this mutation considering its important role in AChE. The Y148F data, along with the E217M and F354 results, all reinforce the expectation that E3 has substantially diverged from the p1 (anionic) site found in the cholinesterases. The Y148F data further suggest that this substitution does not contribute to the OP hydrolase activity and OP resistance which Claudianos et al. (1999) found for the Rutgers allele of the E3 ortholog in M. domestica. The same Y148F mutation has also been found in this enzyme in the NG98 and YPER housefly strains (Scott and Zhang, 2003), apparently not associated with resistance. 3.4. Evolutionary implications While only a small number of synthetic active site E3 mutants have been tested, at least two have been identified that are kinetically equivalent or superior to the natural OP resistant variants. There are also indications that other mutations, alone or in combination, might provide further improvements, the most promising involving the W251 series. W251T and W251A both compare favourably with the natural W251L mutation in kinetic parameters relating to malathion resistance. W251T is clearly superior to W251L in dMUP kcat and essentially equivalent in its MCE specificity constant. W251A is equivalent to, or slightly better than, W251L in both activities. W251G is clearly superior to W251L in its MCE kinetics, albeit also clearly inferior in OP hydrolase activities. W251S is slightly better than W251L in MCE kinetics and slightly inferior in OP hydrolase kinetics. The occurrence of mutations equivalent to W251G in A. calandrae (Zhu et al., 1999) and W251S in M. domestica (Claudianos et al., 1999), and both also in E3 variants in African populations of L. cuprina (R.D. Newcomb, pers. comm.), reinforce the potential for further modification of this residue in the evolution of function in this gene. The W251S, W251G and the original W251L replacements all involve single nucleotide changes from W251, whether in the L. cuprina or M. domestica orthologs (Robin et al., 1996; Newcomb et al., 1997; Claudianos et al., 1999). Significantly, however, W251T and W251A each require at least two nucleotide changes, either from W251 or W251L (albeit notably only one from W251S and for W251A also from W251G) so
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that, although of possible greater fitness value in respect of malathion resistance, they may not have yet become available for selection in field populations. Interestingly, F309L also appeared to have some improved OP hydrolase kinetics compared to wild type, but as a double mutant with W251L at least, it was clearly inferior to W251L alone. This would suggest that this particular substitution has little potential as a second site mutation to further augment resistance due to W251L. However, we have only tested one substitution of F309 and the results at least suggest it would be worth investigating others. Recent molecular studies of 41 isogenic L. cuprina strains collected from various Australian and New Zealand populations over the last 20 years (Smythe et al., 2000; Newcomb et al., 2003) have found just seven haplotypes at the E3 locus; two diazinon (G137D) resistant, two malathion (W251L) resistant and three susceptible. Amplification of E3 from seven pinned samples dating back a further 30 years to before the introduction of OP insecticides suggested that one or more of the resistant haplotypes could have been segregating in L. cuprina even before the first use of OP insecticides (Newcomb et al., 2003). These results suggest that OP selection may have exploited pre-existing E3 variants which happened to have some OP hydrolase activities, those variants rapidly sweeping through the population and in the process eliminating many other haplotypes Whilst the locus is now relatively lacking in depauperate of variation, our work suggests that there is scope for second site changes to occur that could augment resistance. Significantly, Smythe et al. (2000) and Newcomb et al. (2003) found that seven of the 41 isogenic strains they studied showed a different sort of second site mutation augmenting resistance, namely a duplication of the locus to capture both a W251L-encoding and a G137D-encoding allele on the one chromosome. This suggests that there is still some selective premium on E3 enzymes with improved OP detoxification abilities.
Acknowledgements This research (including support towards ALD’s secondment to CSIRO Entomology) was funded by The Australian Cotton and Rural Industries Research and Development Corporations, Horticulture Australia Limited, Orica Australia Pty Ltd and the CRC for Sustainable Rice Production, which also provided a PhD scholarship to RH. ALD also received a travelling fellowship from the Royal Society of London.
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