Heidari R, Devonshire A L, Campbell B E, Bell K L, Dorrian S J, Oakeshott J G, Russell R J
CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia.
Insect Biochem Mol Biol. 2004 Apr;34(4):353-63. doi: 10.1016/j.ibmb.2004.01.001.
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 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.
绿蝇（Lucilia cuprina）对有机磷（OP）杀虫剂产生抗性是由于LcalphaE7基因发生突变，该基因编码羧酸酯酶E3，这些突变增强了该酶水解杀虫剂的能力。有两种突变是自然发生的，一种是酯酶氧负离子洞中的G137D，另一种是酰基结合口袋中的W251L。先前对克隆基因进行的这些修饰的体外诱变和表达已证实了它们的功能意义。G137D分别使磷酸二乙酯和磷酸二甲酯的水解增强了55倍和33倍。W251L同样增加了磷酸二甲酯的水解，但对磷酸二乙酯同系物仅增强了10倍；然而，与G137D不同的是，它还保留了水解马拉硫磷离去基团中羧酸酯（马拉硫磷羧酸酯酶，MCE）的能力，从而赋予对该化合物的强抗性。在本研究中，我们用其他预期会影响酶效率的氨基酸替换了这些以及附近的氨基酸。将G137替换为谷氨酸或组氨酸在提高OP水解酶活性方面不如天冬氨酸有效，并且与G137D一样，它降低了MCE活性，主要是通过增加Km实现的。将W251替换为其他较小残基的各种替代对OP水解酶和MCE活性的影响与W251L大致相似，但至少有两种在与马拉硫磷抗性相关的动力学参数方面在数量上更好。其中一种，W251G，天然存在于一种对马拉硫磷具有抗性的膜翅目寄生蜂中，使MCE活性提高了20倍以上。催化裂隙底部附近其他位点的突变通常会降低OP水解酶和MCE活性，但有一种，F309L，也使OP水解酶活性有了一些提高。结合对酶 - 底物相互作用可能的空间效应以及该基因的未来进化对结果进行了讨论。
