Isolation and expression of a cDNA clone encoding human kynureninase

被引:39
作者
AlberatiGiani, D
Buchli, R
Malherbe, P
Broger, C
Lang, G
Kohler, C
Lahm, HW
Cesura, AM
机构
[1] F HOFFMANN LA ROCHE & CO LTD,DIV PHARMA,PRECLIN RES,NERVOUS SYST DIS,CH-4070 BASEL,SWITZERLAND
[2] F HOFFMANN LA ROCHE & CO LTD,COMPUTAT CHEM DEPT,CH-4002 BASEL,SWITZERLAND
[3] F HOFFMANN LA ROCHE & CO LTD,GENE TECHNOL,CH-4002 BASEL,SWITZERLAND
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1996年 / 239卷 / 02期
关键词
kynureninase; kynurenine pathway; tryptophan metabolism; cysteine-S-conjugate beta-lyase; pyridoxal-5'-phosphate-dependent enzymes;
D O I
10.1111/j.1432-1033.1996.0460u.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 G(2)) 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 = 52 357 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 approximate to 2.0-kb mRNA species in all tissues tested. A second mRNA species (approximate to 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 mu M and 13.2 +/- 2.0 mu M, respectively.
引用
收藏
页码:460 / 468
页数:9
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