Isolation and Expression of a cDNA Clone Encoding Human Kynureninase

Abstract

Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyridoxal-P)-depe11dent 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,) 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 Sarcharomyces 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 K,,, values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 -C 37 pM and 13.2 t 2.0 pM, respectively.

Enzymology of Nad + Synthesis

“…Furthermore, inspection of the human kynureninase amino acid sequence showed the existence of a consensus sequence at the pyridoxal phosphate binding site. The recombinant protein major molecular and kinetic properties essentially resembled those already described for the mammalian enzyme (Alberati-Giani et al, 1996b).…”

“…The most extensive studies on this enzyme have been conducted in rats (Kaway et al, 1988). Kinetic measurements indicate a clear preference of the enzyme for L-3-hydroxykynurenine respect to the other substrate L-kynurenine (Takeuchi et al, 1980;Alberati-Giani et al, 1996b). From rat liver 3-hydroxylkynureninase amino acid sequence, it has been possible to identify the coenzyme pyridoxal phosphate-binding residue, as LYS*'~.…”

“…Furthermore, it has been observed that interferony stimulates kynureninase in murine macrophages (Alberati-Giani et al, 1996a). This interest, which is beyond the scope of the present discussion, prompted researchers to launch an in-depth investigation of human kynureninase, whose cDNA has been isolated and expressed (Alberati-Giani et al, 1996b). In analyses of the predicted amino acid sequence, the human enzyme shows a 84% amino acid identity and 92% similarity with the sequence of the rat enzyme (Takeuchi et al, 1995).…”

Enzymology of NAD+ homeostasis in man

“…Similar to IDO and K3H, human kynureninase is significantly activated by bacterial lipopolysaccharides and proinflammatory cytokines in human monocyte-derived macrophages [61]. The human cDNA codes for a protein of 456 residues, with a M r of 52,400 [70,71]. Northern blot analyses performed on different human organs and tissues reveal that the enzyme is ubiquitous, being mostly expressed in the liver, placenta and lung [70].…”

Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

“…In particular, the exogenous administration of 3-HK in adult insects profoundly affects locomotive activity and can culminate in irreversible paralysis and death (15,16). The biochemical description of the kynurenine pathway from a number of vertebrates revealed that 3-HK can undergo two alternative fates: either (i) it is hydrolyzed by a specific kynureninase to alanine and 3-hydroxyanthranilic acid, feeding the de novo synthesis of the essential cofactor NAD (17), or (ii) it can be directly converted into the more chemically stable compound xanthurenic acid (XA) by irreversible transamination. In rodents and humans, this latter function is provided by the same kynurenine aminotransferase (KAT) isozymes that are responsible for the pyridoxal 5Ј-phosphate (PLP)-dependent transformation of L-KYN into the neuroprotective and anticonvulsant NMDA receptor antagonist kynurenic acid (KA) (18)(19)(20).…”