Abstract
Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs.
Highlights
The reemergence of malaria, a disease transmitted to humans by mosquitoes, is associated with vector resistance in many tropical countries due to the persistent use of pyrethroid insecticides [1,2].The insecticide resistance has been reported in Anopheles minimus mosquito, one of the malaria primary vectors in Thailand [3]
W678A and W678H increased nicotinamide adenosine diphosphate reduced form (NADPH) binding affinity by two- to three-fold compared to wild-type and L86F/L219F mutant A. minimus cytochrome P450 oxidoreductase (CYPOR) enzyme (AnCYPOR) enzymes, but not that to NADH (Table 2)
Comparative modeling of AnCYPOR structure demonstrated differences in topology arrangement of AnCYPOR enzyme compared to rat CYPOR structure
Summary
The reemergence of malaria, a disease transmitted to humans by mosquitoes, is associated with vector resistance in many tropical countries due to the persistent use of pyrethroid insecticides [1,2]. The purified flAnCYPOR enzyme can support baculovirus-expressed CYP6AA3 and CYP6P7 to metabolize pyrethroid insecticides and benzyloxyresorufin fluorescent substrate in vitro, but the purified enzyme is prone to lose FAD and FMN cofactors compared to rat CYPOR [7,8,9,14,15]. Unlike rat and house fly CYPORs, the open/highly flexible structure of AnCYPOR could explain that AnCYPOR loosely binds flavin cofactors and can be reconstituted with exogenous FMN and FAD in vitro [14,15,16,17]. In this context, why the A. minimus CYPOR is enormously different from rat CYPOR in stability, activity in its native form and in the ease in which flavin cofactors are lost remains unanswered. Our results have contributed to an understanding of the typical nature of mosquito CYPOR compared to rat CYPOR, and its efficacy in electron transfer in mosquito P450-mediated metabolisms
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