Bayer AG, Crop Science Division, Alfred Nobel Str. 50, D-40789 Monheim am Rhein, Germany; Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom.
Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom.
Pestic Biochem Physiol. 2022 Mar;182:105051. doi: 10.1016/j.pestbp.2022.105051. Epub 2022 Feb 4.
Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 - tested at different multiplicity of infection (MOI) ratios - resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4'OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin.
细胞色素 P450 单加氧酶（P450s）是研究较为深入的一类酶，可催化昆虫中外源物质的氧化代谢，包括蚊子。在农业和公共卫生害虫（如疟蚊）中，这些酶的复制和上调通常会导致杀虫剂代谢增强，从而产生抗药性。在实验室品系冈比亚按蚊 FUMOZ-R 中，复制的 P450 酶 CYP6P9a 和 CYP6P9b 高度上调，并被证明可赋予拟除虫菊酯抗性。微粒体 P450 活性受 NADPH 细胞色素 P450 氧化还原酶（CPR）调控，后者是电子转移所必需的，而细胞色素 b（CYB5）对昆虫 P450 活性的调节作用则不太清楚。在之前的研究中，使用大肠杆菌表达系统，将 CYP6P9a 和 CYP6P9b 与按蚊 CPR 串联重组表达，并向反应混合物中添加 CYB5 以增强活性。然而，当与 CYP6P9a 和 CYP6P9b 结合时，CYB5 对底物转化的精确作用仍未得到充分研究，因此本研究的一个目标是解决这一知识空白。与 CYP6P9 变体不同，与敏感的 FANG 品系相比，在对拟除虫菊酯具有抗性的 FUMOZ-R 品系中，CYB5 和 CPR 的表达水平均未上调，这表明这些基因在 FUMOZ-R 对拟除虫菊酯的抗性中没有直接的调节作用。在这里，我们首次使用杆状病毒表达系统，在 High-5 昆虫细胞中从致倦库蚊重组表达 CYP6P9a 和 CYP6P9b。用来自冈比亚按蚊或致倦库蚊的 CPR 共表达每种酶，并未显示出催化能力的显著差异。然而，用不同感染复数（MOI）比例的致倦库蚊 CYB5 共表达，在荧光测定中导致香豆素底物的代谢明显增加。使用最活跃的底物 7-苄氧基甲氧基-4-三氟甲基香豆素（BOMFC）的米氏-门坦动力学分析证实了这一点。我们观察到在反应混合物中添加人 CYB5 也会使香豆素底物的转化率增加。最后，我们通过 UPLC-MS/MS 分析比较了重组表达的 CYP6P9a 和 CYP6P9b 与 CYB5 共表达（或添加）时，溴氰菊酯的消耗率和 4'OH-溴氰菊酯的形成，未检测到溴氰菊酯代谢程度的差异。我们的结果表明，在昆虫细胞中重组表达的 CYP6P9 变体与 CYB5 共表达（或添加）可显著增强其氧化香豆素的代谢能力，但对溴氰菊酯则不然。
