Testing the evolvability of an insect carboxylesterase for the detoxification of synthetic pyrethroid insecticides.
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.
CSIRO Ecosystem Sciences, Canberra, ACT 2601, Australia. chris.coppin@csiro.au
Chris W
CW
Coppin
Colin J
CJ
Jackson
Tara
T
Sutherland
Peter J
PJ
Hart
Alan L
AL
Devonshire
Robyn J
RJ
Russell
John G
JG
Oakeshott
D019943:Amino Acid Substitution; D000818:Animals; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism; D004175:Diptera / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli; D019143:Evolution, Molecular; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007305:Insecticide Resistance; D007306:Insecticides; D009154:Mutation; D009570:Nitriles; D017434:Protein Structure, Tertiary; D011722:Pyrethrins; D011995:Recombination, Genetic; D013237:Stereoisomerism
D019476:Insect Proteins; D007306:Insecticides; D009570:Nitriles; D011722:Pyrethrins; C017160:cypermethrin; C017180:decamethrin; D043182:Carboxylesterase; C017690:fenvalerate