cDNA cloning, functional expression and characterization of kynurenine 3-hydroxylase of Anopheles stephensi (Diptera: Culicidae).
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.
Hirai
M
M
Department of Medical Zoology, Jichi Medical School, Minamikawachi, Tochigi, Japan. mhirai@ms.jichi.ac.jp
Kiuchi
M
M
Wang
J
J
Ishii
A
A
Matsuoka
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D050601:Kynurenine 3-Monooxygenase; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D018076:DNA, Complementary; D006899:Mixed Function Oxygenases; D050601:Kynurenine 3-Monooxygenase