Biochemical Characteristics And Functions Of PPO6 And PPO10 From Aedes Aegypti

Mosquitoes are important ectoparasites and the number one vector of epidemic diseases.In taxonomy,mosquitoes belong to Diptera insects.Insect prophenoloxidases(PPOs)are a type ? copper protein that catalyzes the synthesis of melanin,involved in the formation of cuticles and eggshells,wound healing and encapsulation,and fighting invasive pathogens by oxidizing active free radicals.Therefore,the study of prophenoloxidase in mosquitoes is helpful to understand the mechanism of mosquito transmission and defense against pathogens.Phenoloxidase(PO)in insects usually exists in the form of prophenoloxidase(PPO)and then is activated by protease hydrolysis.Compared with other insects,mosquitoes have more PPO genes,but the specific biochemical functions of these PPOs are still unclear.It is expected that the functional mechanism of PPO of Aedes aegypti could be further explored,and the experimental basis for classification,nomenclature and annotation of PPO of insects could be provided.The phylogenetic tree was constructed by MAGE 5,and the species distribution analysis confirmed that Ae.aegypti contained a prophenoloxidase PP06 closely related to non-mosquito insect PPO,while the rest of PPOs were mosquito specific.Two genes,PPO6 and PPO10,were selected for their gene expression study.The highest expression of PP06 gene was found in the male mosquitoes midgut and the first instar larvae,and the highest expression of PPO10 gene was found in the female mosquitoes head and the first instar larvae.Then the two proteins were expressed in E.coli cells.The enzyme activity of the expressed proteins was determined and the enzyme kinetics was calculated using dopamine,L-DOPA and tyrosine as substrates.The results showed that PP06 only reacted with diphenolic compounds,and PPO10 reacted with both monophenolic and diphenolic compounds.The three-dimensional models of PP06 and PPO10 were constructed by SWISS-MODEL,and the PPO structures were docked with tyrosine substrate by AutoDock.By comparing its structural characteristics of PP06 and PPO10,it was indicated that PPO6 could not react with tyrosine because of the blocking of Glu-216 in PP06.Asp-21 8 of PPO 10 might be the key residue for tyrosine interaction.