Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase.
Spontaneous oxidation of 3-hydroxykynureine (3-HK), a metabolic intermediate of the tryptophan degradation pathway, elicits a remarkable oxidative stress response in animal tissues. In the yellow fever mosquito Aedes aegypti the excess of this toxic metabolic intermediate is efficiently removed by a specific 3-HK transaminase, which converts 3-HK into the more stable compound xanthurenic acid. In anopheline mosquitoes transmitting malaria, xanthurenic acid plays an important role in Plasmodium gametocyte maturation and fertility. Using the sequence information provided by the Anopheles gambiae genome and available ESTs, we adopted a PCR-based approach to isolate a 3-HK transaminase coding sequence from the main human malaria vector A. gambiae. Tissue and developmental expression analysis revealed an almost ubiquitary profile, which is in agreement with the physiological role of the enzyme in mosquito development and 3-HK detoxification. A high yield procedure for the expression and purification of a fully active recombinant version of the protein has been developed. Recombinant A. gambiae 3-HK transaminase is a dimeric pyridoxal 5'-phosphate dependent enzyme, showing an optimum pH of 7.8 and a comparable catalytic efficiency for both 3-HK and its immediate catabolic precursor kynurenine. This study may be useful for the identification of 3-HK transaminase inhibitors of potential interest as malaria transmission-blocking drugs or effective insecticides.
Rossi
Franca
F
DiSCAFF, University of Piemonte Orientale Amedeo Avogadro, Novara, Italy.
Lombardo
Fabrizio
F
Paglino
Alessandra
A
Cassani
Camilla
C
Miglio
Gianluca
G
Arcà
Bruno
B
Rizzi
Menico
M
D000595:Amino Acid Sequence; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D007737:Kynurenine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014977:Xanthurenates / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014977:Xanthurenates; D007737:Kynurenine; C028330:xanthurenic acid; D000637:Transaminases