Isolation, characterization, and functional expression of kynurenine aminotransferase cDNA from the yellow fever mosquito, Aedes aegypti(1).
This study describes the molecular and biochemical characterization of kynurenine aminotransferase (KAT) from Aedes aegypti mosquitoes. Through screening an A. aegypti larval cDNA library, a 1695-bp full-length cDNA clone with a 1434-bp open reading frame (ORF) was isolated. Its deduced amino acid sequence consists of 477 amino acid residues with a predicted molecular mass of 53,490 and the amino acid sequence shares 47 and 42% identity with KATs from Homo sapiens and Rattus norvegicus, respectively. This putative A. aegypti KAT (AeKAT) has four potential N-glycosylation sites (Asn-Xxx-Trp/Ser) and a typical mitochondrial leader sequence consisting of 49 amino acids at its NH(2)-terminus with a putative cleavage site between Met-49 and Ser-50. A consensus pyridoxal 5'-phosphate (PLP) binding domain (Gly-Ser-Ala-Gly-Lys-Thr-Phe-Ser) is present in the central region of the ORF. Using a baculovirus/insect cell expression system, a full-length AeKAT and a truncated AeKAT, lacking the mitochondrial leader sequence, were expressed. The full-length AeKAT expressed in Sf9 insect cells is insoluble and has no detectable KAT activity, but the truncated AeKAT is soluble and capable of catalyzing the transamination of kynurenine to kynurenic acid in the presence of pyruvate as an amino group acceptor. However, the expressed truncated protein is not active to 3-hydroxykynurenine. Northern analysis indicates that transcription of the AeKAT occurs at all stages during mosquito development, but higher levels of mRNA are observed during the pupal and adult stages. Results indicate that a specific KAT is present in mosquitoes and the enzyme may play an important physiological role in A. aegypti.
Department of Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.
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Fang
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Han
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Li
D000330:Aedes / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017344:Genes, Insect; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase