Cloning and recombinant expression of rat and human kynureninase.
Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase 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 kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.
Toma
S
S
Bioscience Center, Pharmacia and Upjohn, Nerviano, Italy.
Nakamura
M
M
Toné
S
S
Okuno
E
E
Kido
R
R
Breton
J
J
Avanzi
N
N
Cozzi
L
L
Speciale
C
C
Mostardini
M
M
Gatti
S
S
Benatti
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D013437:Carbon-Sulfur Lyases; D003001:Cloning, Molecular; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007737:Kynurenine / Q000031:analogs & derivatives / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008190:Lyases / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D017421:Sequence Analysis; D014162:Transfection / Q000235:genetics
D012333:RNA, Messenger; C005045:3-hydroxykynurenine; D007737:Kynurenine; D006867:Hydrolases; C021480:kynureninase; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C019969:S-alkylcysteine lyase