期刊
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY
卷 34, 期 4, 页码 353-363出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.ibmb.2004.01.001
关键词
Lucilia cuprina; esterase; kinetics; mutagenesis; organophosphorus insecticides; resistance
Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, 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 NICE activity, primarily through increases in K-m. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L oil 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 NICE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and NICE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects oil enzyme-substrate interactions and future evolution of this gene. (C) 2004 Elsevier Ltd. All rights reserved.
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