Isolation and Expression of a cDNA Clone Encoding Human Kynureninase

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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 ≈2.0-kb mRNA species in all tissues tested. A second mRNA species (≈2.6 kb) was also detected in some tissues. After transfection of HEK-293 cells with the cDNA coding for kynureninase, the Km, values of l-kynurenine and dl-3-hydroxykynurenine for the recombinant enzyme were 671±37 μM and 13.2±2.0 μM, respectively.