Cloning and Functional Expression of a Soluble Form of Kynurenine/α -Aminoadipate Aminotransferase from Rat Kidney

Abstract

Several aminotransferases with kynurenine aminotransferase (KAT) activity are able to convert L-kynurenine into kynurenic acid, a putative endogenous modulator of glutamatergic neurotransmission. In the rat, one of the described KAT isoforms has been found to correspond to glutamine transaminase K. In addition, rat kidney ␣-aminoadipate aminotransferase (AadAT) also shows KAT activity. In this report, we describe the isolation of a cDNA clone encoding the soluble form of this aminotransferase isoenzyme from rat (KAT/AadAT). Degenerate oligonucleotides were designed from the amino acid sequences of rat kidney KAT/AadAT tryptic peptides for use as primers for reverse transcriptionpolymerase chain reaction of rat kidney RNA. The resulting polymerase chain reaction fragment was used to screen a rat kidney cDNA library and to isolate a cDNA clone encoding KAT/AadAT. Analysis of the combined DNA sequences indicated the presence of a single 1275-base pair open reading frame coding for a soluble protein of 425 amino acid residues. KAT/AadAT appears to be structurally homologous to aspartate aminotransferase in the pyridoxal 5-phosphate binding domain. RNA blot analysis of rat tissues, including brain, revealed a single species of KAT/AadAT mRNA of ϳ2.1 kilobases. HEK-293 cells transfected with the KAT/AadAT cDNA exhibited both KAT and AadAT activities with enzymatic properties similar to those reported for the rat native protein.

Biochemical and Phenotypic Abnormalities in Kynurenine Aminotransferase II-Deficient Mice

“…In the rat liver, four enzymes are capable of irreversibly converting kynurenine to KYNA, but their relative contributions to KYNA synthesis in vivo are unknown (32). Rat and human brain contain two KATs, arbitrarily termed KAT I and KAT II, which are identical to two of the known peripheral KATs, glutamine aminotransferase K and L-␣-aminoadipate aminotransferase, respectively (6,20,41). In contrast to KAT I, KAT II has a physiological pH optimum and lacks substrate competition by glutamine, tryptophan, phenylalanine, and other amino acids which are present in the brain in high concentrations.…”

Crystal Structure of Human Kynurenine Aminotransferase II

“…The identity of rat KAT-II to aminoadipate aminotransferase was confirmed 30 years ago (51), and the KAT-II gene was isolated by RT-PCR from rat kidney (52) or EST assembly from mouse (16) and human (53). Recombinant mouse KAT-II is active to both aminoadipate and kynurenine and prefers ketoglutarate as an amino group receptor (52). Recombinant hKAT-II has shown activity toward aminoadipate and ketoglutarate (53), and together with our results, we can conclude that hKAT-II is also active to both kynurenine and aminoadipate.…”

Isolation and Expression of a cDNA Clone Encoding Human Kynureninase

“…For instance, as mentioned above, kynureninase also displays cysteine-S-conjugate P-lyase activity. Notably, a kynurenine aminotransferase/glutamine transaminase K form (Perry et al, 1993;Alberati-Giani et al, 1995), an a-family protein structurally homologous to aspartate aminotransferase (Buchli et al, 1995), also shows p-lyase activity. No kynurenine amino-transferase activity was detected in HEK-293 cells transfected with the human kynureninase cDNA construct after incubation with L-kynurenine and pyruvate or 2-oxoglutarate as amino acceptors.…”