CLONING AND RECOMBINANT EXPRESSION OF RAT AND HUMAN KYNURENINASE

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the bios ynthesis of NAD cofactors from tryptophan through the kynurenine pathw ay. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The ra t kynureninase cDNA, isolated by means of reverse-transcribed polymera se chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynurenina se was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cD NAs. The K-m values of the rat enzyme, for L-kynurenine and DL-3-hydro xykynurenine, were 440 +/- 20 mu M and 32 + 5 mu M and of the human en zyme 440 +/- 20 mu M and 49 +/- 6 mu M, respectively. Interestingly, C OS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity. (C) 1997 Federation of European Biochemical Societies.