ISOLATION AND EXPRESSION OF A CDNA CLONE ENCODING HUMAN KYNURENINASE

Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyrid oxal-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 fro m the amino acid sequences of peptides from rat liver kynureninase, we re used as primers for reverse-transcription PCR of rat kidney RNA. Th e 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 s howed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical mo lecular mass = 52 357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat en zyme and to a Saccharomyces cerevisiae gene product putatively ascribe d to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P-binding site consensus sequenc e assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including b rain, 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 wit h 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.