Analysis of the wild-type and mutant genes encoding the enzyme kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti.
Kynurenine 3-monooxygenase (KMO) catalyses the hydroxylation of kynurenine to 3-hydroxykynurenine. KMO has a key role in tryptophan catabolism and synthesis of ommochrome pigments in mosquitoes. The gene encoding this enzyme in the yellow fever mosquito, Aedes aegypti, is called kynurenine hydroxylase (kh) and a mutant allele that produces white eyes has been designated khw. A number of cDNA clones representative of wild-type and mutant genes were isolated. Sequence analyses of the wild-type and mutant cDNAs revealed a deletion of 162 nucleotides in the mutant gene near the 3'-end of the deduced coding region. RT-PCR analyses confirm the transcription of a truncated mRNA in the mutant strain. The in-frame deletion results in a loss of 54 amino acids, which disrupts a major alpha-helix and which probably accounts for the loss of activity of the enzyme. Recombinant Ae. aegypti KMO showed high substrate specificity for kynurenine with optimum activity at 40 degrees C and pH = 7.5. Kinetic parameters and inhibition of KMO activity by Cl- and pyridoxal-5-phosphate were determined.
Han
Q
Q
Department of Pathobiology, University of Illinois at Urbana-Champaign, IL, USA.
Calvo
E
E
Marinotti
O
O
Fang
J
J
Rizzi
M
M
James
A A
AA
Li
J
J
D000330:Aedes / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D020869:Gene Expression Profiling; D007700:Kinetics; D050601:Kynurenine 3-Monooxygenase; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D018076:DNA, Complementary; D006899:Mixed Function Oxygenases; D050601:Kynurenine 3-Monooxygenase