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cDNA cloning, functional expression and characterization of kynurenine 3‐hydroxylase of Anopheles stephensi (Diptera: Culicidae)
Author(s) -
Hirai M.,
Kiuchi M.,
Wang J.,
Ishii A.,
Matsuoka H.
Publication year - 2002
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1046/j.1365-2583.2002.00358.x
Subject(s) - anopheles stephensi , biology , xanthurenic acid , complementary dna , microbiology and biotechnology , plasmodium knowlesi , plasmodium falciparum , recombinant dna , biochemistry , gene , aedes aegypti , amino acid , tryptophan , plasmodium vivax , malaria , larva , botany , immunology
Kynurenine 3‐hydroxylase (K3H) is a NADPH‐dependent flavin monooxygenase involved in the tryptophan pathway. Xanthurenic acid (XA) is a metabolite of this pathway and has recently been identified as a gamete activating factor (GAF) of the malarial parasite. We cloned K3H cDNA from Anopheles stephensi (AsK3H), because anopheline mosquitoes are a vector of the human malaria parasite, Plasmodium falciparum and the catalytic function of AsK3H in XA production. Recombinant AsK3H protein was expressed in Sf‐9 cells using the baculovirus system and its enzymatic properties were characterized. The specific activities of crude cell lysate and affinity purified protein were 94.9 ± 6.2 and 865.6 ± 10.5 nmol/min/mg protein, respectively. The optimum pH of AsK3H was 7.0. Analysis of AsK3H gene expression using RT‐PCR revealed that AsK3H was constitutively expressed in egg, larva, pupa and adult.