|
type |
Journal Article |
authors |
Alberati-Giani, D.; Buchli, R.; Malherbe, P.; Broger, C.; Lang, G.; Kohler, C.; Lahm, H. W.; Cesura, A. M. |
title |
Isolation and expression of a cDNA clone encoding human kynureninase |
journal |
Eur J Biochem |
Activity |
3.7.1.3 |
ui |
96314506 |
year |
(1996) |
volume |
239 |
number |
2 |
pages |
460-8. |
| |
keywords |
Amino Acid Sequence |
abstract |
Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate- (pyridoxal-P)-dependent enzyme, catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse-transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G2) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P- binding site consensus sequence assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an approximately 2.0-kb mRNA species in all tissues tested. A second mRNA species (approximately 2.6 kb) was also detected in some tissues. After transfection of HEK-293 cells with the cDNA coding for kynureninase, the K(m) values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 +/- 37 microM and 13.2 +/- 2.0 microM, respectively. |
last changed |
2002/11/12 16:17 |
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