Initial characterization of a recombinant kynureninase from Trypanosoma cruzi identified from an EST database.
Kynureninase has been described in bacteria, fungi and animals as an enzyme involved in the catabolic degradation pathway of l-tryptophan. This pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the hydrolytic cleavage of l-kynurenine and 3-hydroxy-l-kynurenine to yield l-alanine and either anthranilic or 3-hydroxyanthranilic acid, respectively. We identified a putative kynureninase gene from a Trypanosoma cruzi project aiming at the structural and functional characterization of more than 100 proteins differentially expressed during metacyclogenesis. This gene encodes a protein similar in size and sequence to kynureninases from other sources. This open reading frame was cloned and the recombinant enzyme was overexpressed. Recombinant T. cruzi kynureninase was purified to homogeneity and its identity was confirmed by mass spectrometry. The apparent molecular mass of the native T. cruzi kynureninase was estimated by gel filtration, suggesting that the protein is a homodimer. Circular dichroism spectrum indicated a mixture of alpha-helix and beta-sheet structure, expected for an aminotransferase fold. l-kynurenine, preferentially hydrolyzed by prokaryotic inducible kynureninases, and 3-hydroxy-l-kynurenine, the preferred substrate in fungi and vertebrates, are both catabolized equally well by T. cruzi kynureninase. Further experimental assays will be performed to fully understand the importance of this enzyme for T. cruzi metabolism.
Ecco
Gabriela
G
Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
Vernal
Javier
J
Razzera
Guilherme
G
Tavares
Carolina
C
Serpa
Viviane Isabel
VI
Arias
Santiago
S
Marchini
Fabricio Klerynton
FK
Krieger
Marco Aurélio
MA
Goldenberg
Samuel
S
Terenzi
Hernán
H
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D030562:Databases, Protein; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016364:Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000201:enzymology*
D011994:Recombinant Proteins; D006867:Hydrolases; C021480:kynureninase