Expression and biochemical properties of a recombinant acetylcholinesterase 1 of the sand fly, Phlebotomus papatasi (Scopoli), insensitive to organophosphate inhibition

Phlebotomine sand flies are small hematophagous flies present throughout tropical and subtropical areas of the world and are vectors of human and zoonotic leishmaniases.   Human cutaneous leishmaniasis is a debilitating disease presenting major problems for military operations in the Middle East, and people living in areas endemic to phlebotomine sand flies.  We recently identified and expressed recombinant acetylcholinesterase 1 (rPpAChE1) of Phlebotomus papatasi, one of the principal vectors of human cutaneous leishmaniasis in the Eastern Hemisphere.  The enzyme shared considerable sequence identity with AChE of Lutzomyia spp., the New World Sand Fly, that exists from Argentina to the southern United States.  It was hypothesized that an altered PpAChE1 containing a single amino acid substitution corresponding to that responsible for high level organophosphate insensitivity in AChE1 of mosquitoes may generate a rPpAChE1 insensitive to organophosphate inhibition.  We conducted targeted mutagenesis to generate a cDNA containing the desired codon substitution and expressed the recombinant altered enzyme in a baculovirus expression system.  Biochemical properties of the altered rPpAChE1 containing a substitution of serine for glycine at amino acid position 256 (GenBank accession AFP20868) were quite different from the wild type enzyme, including a significant increase in the Michaelis–Menten constant (K_m) reflecting increased concentration of substrate required to achieve maximal velocity, and 500-1000 fold decreased sensitivity to inhibition by eserine or oxon forms of organophosphates. 

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