Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):10177-82. doi: 10.1073/pnas.1304097110. Epub 2013 Jun 3.
Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10(6) M(-1) s(-1)) indicative of a natural substrate.
昆虫羧酸酯酶来自α酯酶基因簇，例如来自澳大利亚绵羊蕈蝇（Lucilia cuprina）的αE7（也称为 E3）（LcαE7），在脂质代谢中发挥重要的生理作用，并与有机磷（OP）杀虫剂的解毒作用有关。尽管 OP 对农业和昆虫传播疾病的传播非常重要，但α-羧基酯酶赋予昆虫对 OP 抗性的分子基础仍知之甚少。在这项工作中，我们使用实验室进化来提高 LcαE7 的热稳定性，从而允许其在大肠杆菌中过表达并进行结构测定。晶体结构揭示了一个典型的α/β水解酶折叠，与 OP 的主要靶标（乙酰胆碱酯酶）非常相似，并且具有独特的 N 末端α-螺旋，用作膜锚。将 LcαE7 晶体浸泡在 OP 中，捕获了 LcαE7 磷酸化状态的晶体学快照，这允许与乙酰胆碱酯酶进行比较，并合理化其保护昆虫免受 OP 影响的能力。最后，检查 LcαE7 的活性位点揭示了一个不对称的疏水性底物结合腔，非常适合脂肪酸甲酯，该酶以特异性常数（∼10（6）M（-1）s（-1））水解该酶，表明其为天然底物。
