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 α-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 transcription-polymerase 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 ∼2.1 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. 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 α-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 transcription-polymerase 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 ∼2.1 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. INTRODUCTIONKynurenine aminotransferase (EC 2.6.1.7, KAT 1The abbreviations used are: KATkynurenine aminotransferaseAadATα-aminoadipate aminotransferaseGTKglutamine transaminase KAATaspartate aminotransferasePLPpyridoxal 5′-phosphateRTreverse transcriptionPCRpolymerase chain reactionbpbase pair(s)HPLChigh performance liquid chromatographyFPLCfast protein liquid chromatography. ) catalyzes the irreversible transamination of the L-tryptophan metabolite L-kynurenine to form kynurenic acid. Due to an overlapping substrate specificity, multiple forms of pyridoxal 5′-phosphate (PLP)-dependent aminotransferases are apparently able to catalyze this reaction(1Deshmukh D.R. Mungre S.M. Biochem. J. 1989; 261: 761-768Google Scholar). Differences probably exist in the various tissues and among different animal species regarding which enzyme form is predominantly responsible for the biosynthesis of kynurenic acid. A soluble form of KAT has recently been identified and cloned from rat(2Mosca M. Cozzi L. Breton J. Speciale C. Okuno E. Schwarcz R. Benatti L. FEBS Lett. 1994; 353: 21-24Google Scholar, 3Alberati-Giani D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar), and it was found that the amino acid sequence of this pyruvate-preferring form of KAT is identical to that reported for rat kidney cysteine S-conjugate β-lyase(4Perry S.J. Schofield M.A. MacFarlaine M. Lock E.A. King L.J. Gibson G.G. Goldfarb P. Mol. Pharmacol. 1993; 43: 660-665Google Scholar), also referred to as glutamine transaminase K (GTK, EC 2.6.1.64)(5Cooper A.J.L. Adv. Pharmacol. 1994; 27: 71-113Google Scholar). The human form of this aminotransferase with KAT, GTK, and β-lyase activity has also been cloned and shown to have 82% amino acid similarity to the rat protein(6Perry S. Harries H. Schofield C. Lock T. King L. Gibson G. Goldfarb P. FEBS Lett. 1995; 360: 277-280Google Scholar).In rat kidney, a 2-oxoglutarate-preferring aminotransferase with KAT activity has also been described. This protein corresponds to α-aminoadipate aminotransferase (AadAT, EC 2.6.1.39), an enzyme involved in the metabolism of lysine (1Deshmukh D.R. Mungre S.M. Biochem. J. 1989; 261: 761-768Google Scholar, 7Tobes M.C. Mason M. J. Biol. Chem. 1977; 252: 4591-4599Google Scholar, 8Takeuchi F. Otsuka H. Shibata Y Biochim. Biophys. Acta. 1983; 743: 323-330Google Scholar, 9Hartline R.A. Methods Enzymol. 1985; 113: 664-672Google Scholar) that catalyzes the reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate.Due to the postulated role of kynurenic acid as a putative endogenous modulator of glutamatergic neurotransmission (for review, see (10Stone T.W. Pharmacol. Rev. 1993; 45: 309-379Google Scholar)), particular attention has recently been devoted to the presence of KAT isoenzymes in cerebral tissues. Kynurenic acid is, in fact, an antagonist at the glycine site of N-methyl-D-aspartate receptors, and increased levels of kynurenic acid may exert a neuroprotective action in some pathological conditions(11Carpenedo R. Chiarugi A. Russi P. Lombardi G. Carlà V. Pellicciari R. Mattoli L. Moroni F. Neuroscience. 1994; 61: 237-244Google Scholar). Two different aminotransferases able to produce kynurenic acid appear to be present in human brain (12Okuno E. Nakamura M. Schwarcz R. Brain Res. 1991; 542: 307-312Google Scholar, 13Schmidt W. Guidetti P. Okuno E. Schwarcz R. Neuroscience. 1993; 55: 177-184Google Scholar, 14Baran H. Okuno E. Kido R. Schwarcz R. J. Neurochem. 1994; 62: 730-738Google Scholar). A cDNA clone from rat brain encoding an aminotransferase with KAT activity has been recently isolated(3Alberati-Giani D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar), and it has been found that this protein corresponds to the rat kidney β-lyase/GTK enzyme cloned by Perry et al.(4Perry S.J. Schofield M.A. MacFarlaine M. Lock E.A. King L.J. Gibson G.G. Goldfarb P. Mol. Pharmacol. 1993; 43: 660-665Google Scholar). Rat brain also contains a mitochondrial form of this aminotransferase, which differs from cytosolic KAT/GTK exclusively for the presence of an NH2-terminal leader peptide, which targets the protein to the mitochondrial matrix(15Malherbe P. Alberati-Giani D. Köhler C. Cesura A.M. FEBS Lett. 1995; 367: 141-144Google Scholar). Whereas it has been claimed that in rats, a single pyruvate-preferring enzyme with KAT activity may be of physiological relevance in the cerebral synthesis of kynurenic acid(16Okuno E. Du F. Ishikawa T. Tsujimoto M. Nakamura M. Schwarcz R. Kido R. Brain Res. 1990; 534: 37-44Google Scholar, 17Okuno E. Schmidt W. Parks D.A. Nakamura M. Schwarcz R. J. Neurochem. 1991; 57: 533-540Google Scholar), whether in rat brain other aminotransferase forms are involved in the biosynthesis of kynurenic acid remains to be established. Noteworthy, the presence of AadAT activity has also been described in rat brain (see (18Huck S. Grass F. Hörtnagl H. J. Neurosci. 1984; 4: 2650-2657Google Scholar) and references therein), therefore suggesting that this aminotransferase may represent a second synthetic enzyme for kynurenic acid in the central nervous system of this animal species.In the present work, we describe the molecular cloning and the functional expression of a soluble aminotransferase from rat kidney displaying both KAT and AadAT activity (KAT/AadAT).MATERIALS AND METHODSEnzymatic Activity DeterminationKAT activity was assayed as described previously(3Alberati-Giani D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar). For routine analysis, aliquots of the enzyme preparation were incubated (1 h at 37°C) in the presence of 1 mML-kynurenine and 1 mM 2-oxoglutarate in a final volume of 200 μl of 150 mM Tris acetate buffer, pH 8.0, containing 70 μM PLP. Kynurenic acid formed was quantified by HPLC with spectrophotometric detection at 330 nm(3Alberati-Giani D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar). 3-Hydroxykynurenine aminotransferase activity was determined using L-3-hydroxykynurenine as substrate and by measuring the production of xanthurenic acid by HPLC with UV detection at 340 nm.AadAT activity was assayed as described in (19Nakatani Y. Fujioka M. Higashino K. Biochim. Biophys. Acta. 1970; 198: 219-228Google Scholar). Briefly, after incubation of the enzyme preparation in the presence of 1.7 mM 2-oxoadipate and 16.7 mML-glutamate, 2-oxoglutarate formed was measured using the glutamate dehydrogenase assay. Aminotransferase activity toward other L-amino acids was measured essentially as described previously(14Baran H. Okuno E. Kido R. Schwarcz R. J. Neurochem. 1994; 62: 730-738Google Scholar).Kinetic constants were calculated by fitting the experimental data to Michaelis-Menten equation using a computer program (Ultrafit, Biosoft).Purification of KAT/AadAT from Rat KidneyRat kidneys were homogenized in 10 mM potassium phosphate buffer, pH 7.4 (containing 150 mM NaCl, 10 mM 2-mercaptoethanol, 50 μM PLP, 0.5 mM EGTA, 0.5 mM phenylmethanesulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 2 μg/ml aprotinin), with an Ultra-Turrax homogenizer. at the was to (1 at and to The protein was by by and by a KAT/AadAT was to a (see to was measured by the protein KAT/AadAT and protein was to a and a P. Köhler C. M. Lang G. Kiefer V. Schwarcz R. Cesura A.M. J. Biol. Chem. 1994; Scholar). of KAT/AadAT with and of the resulting peptides were as described P. Köhler C. M. Lang G. Kiefer V. Schwarcz R. Cesura A.M. J. Biol. Chem. 1994; Scholar, G. J. M. J. S. A. 1989; Scholar). KAT/AadAT tryptic peptides were by HPLC (for the see D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google and to amino acid sequence analysis by of an protein with of RNA was from different rat tissues by the Scholar). RNA was by of Rat KAT/AadAT cDNA by were in 10 mM pH 50 mM mM 1 mM 1 mM 1 mM 1 mM of of 1 of rat kidney and 50 of cloned reverse in a final volume of μl at for was in a final volume of μl containing μl of the reverse 10 mM pH 50 mM 2 mM mM mM mM mM of and of DNA polymerase was at (1 (1 and (1 for Degenerate primers were by final was a The of was from the into the site of the V. M. Malherbe P. S. A. 1991; Scholar), and of cDNA rat kidney cDNA library was from and described R.A. DNA A Scholar). were for h at the were with a rat kidney cDNA for h in mM NaCl, mM mM pH 10 1 mM mM μg/ml μg/ml and μg/ml for at in mM NaCl, mM pH and a in containing the were and to with a of 10 were and an of at the cDNA were from in DNA sequences were determined by the of et F. S. S. A. 1977; Scholar), as for DNA S. from the and and a of primers were DNA sequence analysis, and were using the from various rat tissues were a containing an were by UV for h at the RNA blot was for h with a rat kidney cDNA in mM NaCl, mM pH and 50 μg/ml The were with 2 mM NaCl, mM pH for at and with 1 mM NaCl, mM pH containing for 1 h at of Rat KAT/AadAT in HEK-293 cDNA encoding rat kidney KAT/AadAT was into the site of the expression V. M. Malherbe P. S. A. 1991; Scholar), of the cDNA of the of human kidney cells were transfected with and cDNA as described P. Köhler C. M. Lang G. Kiefer V. Schwarcz R. Cesura A.M. J. Biol. Chem. 1994; Scholar). 2 after the cells were with and at For activity of the the transfected cells were in 0.5 of mM Tris acetate buffer, pH (containing 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 50 μM and homogenized in a at for at aliquots of the were assayed for enzymatic activity as described AND of Rat KAT/AadAT and was from rat kidney the described in The of the enzyme was by measuring KAT activity in the presence of The enzyme also AadAT activity with a activity of this aminotransferase form was from a enzyme with KAT activity and for as In with rat kidney KAT R.A. Methods Enzymol. 1985; 113: 664-672Google Scholar, A. D.R. Biochem. J. 1991; Scholar), indicated of and for KAT/AadAT and (see also D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google The native molecular of KAT/AadAT was in the determined by a and was of of In fact, analysis of the protein a single of in a the amino acid sequence of the protein was to to see was to sequence be that the of the protein was as the of a to protein The amino acid sequences of peptides were after with and of the resulting peptides by The sequence of tryptic peptides and see was were found in the and data of Rat KAT/AadAT the amino acid sequences of the tryptic peptides of rat kidney we were able to the cDNA using Two amino acid sequences from the of peptides and were to synthetic the of the tryptic peptides the protein was and oligonucleotides to the tryptic sequences were A and RNA from rat kidney was using as a and the resulting cDNA was used as a with of and oligonucleotides from the tryptic of one was by for the DNA sequence analysis revealed that the fragment an open reading frame coding for a of amino with the of the amino acid of and with the second of the glutamine of the this cDNA fragment the tryptic peptides and that it was a cDNA for rat of rat kidney KAT/AadAT cDNA fragment by the amino acid sequences of tryptic peptides from rat kidney KAT/AadAT are The of peptides that were for the synthesis of oligonucleotides are and primers to the sequences in A are indicated of and oligonucleotides were used in of rat kidney RNA. from reaction 1 and were by The to a fragment containing the KAT/AadAT protein of Rat KAT/AadAT rat kidney cDNA library from was with the cDNA of rat kidney KAT/AadAT. of revealed 10 with from to The as and were for DNA sequence analysis that were for one of was the coding and it in a in amino acid The and amino acid sequences are shown in Analysis of the combined DNA sequence indicated the presence of a single open reading frame with a at and at with the This open reading frame for a of 425 amino acid with a molecular of is by a containing at and and a in the The is in the sequence which with the sequence found for M. Res. 1984; Scholar). was found of this a coding and amino acid sequence of cloned rat kidney KAT/AadAT. The amino acid sequence of the is shown in the sequence in and is with the are in the to The tryptic peptides from rat kidney KAT/AadAT are The the between is indicated the at at of the sequence with the DNA sequence data using the sequence analysis of indicated that the sequence was and been Analysis of the of the amino acid sequence for KAT/AadAT for with the soluble of the protein. A mitochondrial form of KAT/AadAT has also been F. Otsuka H. Shibata Y Biochim. Biophys. Acta. 1983; 743: 323-330Google Scholar, 9Hartline R.A. Methods Enzymol. 1985; 113: 664-672Google Scholar). those of leader peptides for mitochondrial G. J. Biochem. 1989; Scholar) were in the amino acid sequence of rat KAT/AadAT. whether mitochondrial KAT/AadAT is by a different by a of the form an sequence remains to be established. Rev. Biochem. Scholar) were found in the amino acid sequence of rat kidney KAT/AadAT at and is whether the native protein is with of the protein sequences of KAT/AadAT and KAT/GTK S.J. Schofield M.A. MacFarlaine M. Lock E.A. King L.J. Gibson G.G. Goldfarb P. Mol. Pharmacol. 1993; 43: 660-665Google Scholar) using the program in amino acid with in the and of the amino acid the KAT isoforms similarity of amino acid and similarity were after with other aminotransferases from different including as human E. Y. Nakamura J. M. Kido R. Biochem. J. Scholar), which have been reported to KAT activity. among aminotransferase isoenzymes is by for sequence A multiple sequence of aspartate aminotransferases from various with KAT/AadAT and KAT/GTK that in the central of the sequences are therefore suggesting that the also be In fact, the of amino acid aminotransferases appear to a of structurally homologous that from a single P. J. Biochem. 1993; Scholar). shows an of KAT/AadAT and KAT/GTK with E. aspartate aminotransferase has been in The of E. of an NH2-terminal and and shows the and site of isoenzymes M. J. Mol. Biol. 1994; Scholar) and references The of the of E. to data shows and sequence with KAT/AadAT and the sequence between the KAT isoforms in this is A for this of KAT/AadAT and KAT/GTK was by the amino acids of E. for those of the KAT isoenzymes to the sequence shown in and using the molecular P. K. J. Mol. 1995; Scholar). For both the calculated by the R. D. Scholar) indicated that the of a protein. to the from the sequence of KAT/AadAT and of KAT/GTK correspond to the lysine of the E. which forms an with the In addition, of the involved in the binding of the M. J. Mol. Biol. 1994; Scholar) appear to be in KAT/AadAT and KAT/GTK (see in the and were for molecular This that KAT/AadAT and KAT/GTK in the the site may be different in the by the and and involved in the substrate of KAT/AadAT and KAT/GTK from rat with E. among the acid in KAT/AadAT and KAT/GTK are The involved in binding M. J. Mol. Biol. 1994; Scholar) are The lysine the with is indicated in that the data is used for E. blot analysis of from various rat tissues is shown in In tissues a single species of KAT/AadAT mRNA of was using a cDNA from that the in KAT/AadAT mRNA may be cloned in the Whereas was for mRNA from kidney, a of was also in RNA from brain and cerebral the presence of this enzyme form in cerebral tissues. The found in brain in to tissues is in with the activity of KAT isoenzymes in cerebral tissues (see W. Guidetti P. Okuno E. Schwarcz R. Neuroscience. 1993; 55: 177-184Google The that KAT/AadAT was in the be to the mRNA for this protein in this brain blot of from various rat tissues. The blot was with a cDNA from the as determined by RNA The blot was to for and of Rat KAT/AadAT in HEK-293 and of the that the cDNA for an aminotransferase with both KAT and AadAT the rat KAT/AadAT cDNA was into the expression V. M. Malherbe P. S. A. 1991; Scholar). HEK-293 cells were for KAT AadAT activity. A expression of both KAT and AadAT activities be after of with of the enzymatic activity in the soluble of the activity was after of the cells with the of KAT properties of the enzyme with 2 mM 2-oxoglutarate as amino and for L-kynurenine of mM and The enzyme also L-3-hydroxykynurenine to xanthurenic acid with a similar In the presence of mML-kynurenine as amino the of 2-oxoglutarate was the enzyme was assayed for AadAT the for was with a of experimental the of KAT/AadAT to be similar for both KAT and AadAT as it be from the similar The are in with those found for the rat native D.R. Mungre S.M. Biochem. J. 1989; 261: 761-768Google Scholar, 8Takeuchi F. Otsuka H. Shibata Y Biochim. Biophys. Acta. 1983; 743: 323-330Google Scholar, Y. Fujioka M. Higashino K. Biochim. Biophys. Acta. 1970; 198: 219-228Google KAT activity of the enzyme measured in the presence of various was with 2-oxoglutarate and 2-oxoadipate and with the of this aminotransferase form toward other L-amino aspartate and were found to be for KAT/AadAT. the activities in the presence of amino acids to 10 were and and and of the activity measured with The enzyme enzymatic activity toward the other L-amino acids and The toward amino acids and of the KAT/AadAT in HEK-293 cells was similar to that for the native enzyme from rat in a The of the cDNA clone for this aminotransferase isoenzyme with KAT activity to the of kynurenic acid in as as in the central nervous For in rat brain it appears that at aminotransferases with KAT activity are as described in this and the KAT/GTK which was from cerebral D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar, P. Alberati-Giani D. Köhler C. Cesura A.M. FEBS Lett. 1995; 367: 141-144Google Scholar). Several may the role of the various KAT forms in kynurenic acid as the different constants of for L-kynurenine and the and of in the different of the KAT forms identified in rat, including and are similar to those described for the cerebral KAT forms in E. Nakamura M. Schwarcz R. Brain Res. 1991; 542: 307-312Google Scholar, 13Schmidt W. Guidetti P. Okuno E. Schwarcz R. Neuroscience. 1993; 55: 177-184Google Scholar), therefore the that human KAT forms may be similar to the identified KAT forms from attention has the role of KAT/AadAT in L-tryptophan in metabolism also for in of the lysine the substrate for is a (18Huck S. Grass F. Hörtnagl H. J. Neurosci. 1984; 4: 2650-2657Google Scholar). of the cerebral of this is for the of of and relevance in INTRODUCTIONKynurenine aminotransferase (EC 2.6.1.7, KAT 1The abbreviations used are: KATkynurenine aminotransferaseAadATα-aminoadipate aminotransferaseGTKglutamine transaminase KAATaspartate aminotransferasePLPpyridoxal 5′-phosphateRTreverse transcriptionPCRpolymerase chain reactionbpbase pair(s)HPLChigh performance liquid chromatographyFPLCfast protein liquid chromatography. ) catalyzes the irreversible transamination of the L-tryptophan metabolite L-kynurenine to form kynurenic acid. Due to an overlapping substrate specificity, multiple forms of pyridoxal 5′-phosphate (PLP)-dependent aminotransferases are apparently able to catalyze this reaction(1Deshmukh D.R. Mungre S.M. Biochem. J. 1989; 261: 761-768Google Scholar). Differences probably exist in the various tissues and among different animal species regarding which enzyme form is predominantly responsible for the biosynthesis of kynurenic acid. A soluble form of KAT has recently been identified and cloned from rat(2Mosca M. Cozzi L. Breton J. Speciale C. Okuno E. Schwarcz R. Benatti L. FEBS Lett. 1994; 353: 21-24Google Scholar, 3Alberati-Giani D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar), and it was found that the amino acid sequence of this pyruvate-preferring form of KAT is identical to that reported for rat kidney cysteine S-conjugate β-lyase(4Perry S.J. Schofield M.A. MacFarlaine M. Lock E.A. King L.J. Gibson G.G. Goldfarb P. Mol. Pharmacol. 1993; 43: 660-665Google Scholar), also referred to as glutamine transaminase K (GTK, EC 2.6.1.64)(5Cooper A.J.L. Adv. Pharmacol. 1994; 27: 71-113Google Scholar). The human form of this aminotransferase with KAT, GTK, and β-lyase activity has also been cloned and shown to have 82% amino acid similarity to the rat protein(6Perry S. Harries H. Schofield C. Lock T. King L. Gibson G. Goldfarb P. FEBS Lett. 1995; 360: 277-280Google Scholar).In rat kidney, a 2-oxoglutarate-preferring aminotransferase with KAT activity has also been described. This protein corresponds to α-aminoadipate aminotransferase (AadAT, EC 2.6.1.39), an enzyme involved in the metabolism of lysine (1Deshmukh D.R. Mungre S.M. Biochem. J. 1989; 261: 761-768Google Scholar, 7Tobes M.C. Mason M. J. Biol. Chem. 1977; 252: 4591-4599Google Scholar, 8Takeuchi F. Otsuka H. Shibata Y Biochim. Biophys. Acta. 1983; 743: 323-330Google Scholar, 9Hartline R.A. Methods Enzymol. 1985; 113: 664-672Google Scholar) that catalyzes the reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate.Due to the postulated role of kynurenic acid as a putative endogenous modulator of glutamatergic neurotransmission (for review, see (10Stone T.W. Pharmacol. Rev. 1993; 45: 309-379Google Scholar)), particular attention has recently been devoted to the presence of KAT isoenzymes in cerebral tissues. Kynurenic acid is, in fact, an antagonist at the glycine site of N-methyl-D-aspartate receptors, and increased levels of kynurenic acid may exert a neuroprotective action in some pathological conditions(11Carpenedo R. Chiarugi A. Russi P. Lombardi G. Carlà V. Pellicciari R. Mattoli L. Moroni F. Neuroscience. 1994; 61: 237-244Google Scholar). Two different aminotransferases able to produce kynurenic acid appear to be present in human brain (12Okuno E. Nakamura M. Schwarcz R. Brain Res. 1991; 542: 307-312Google Scholar, 13Schmidt W. Guidetti P. Okuno E. Schwarcz R. Neuroscience. 1993; 55: 177-184Google Scholar, 14Baran H. Okuno E. Kido R. Schwarcz R. J. Neurochem. 1994; 62: 730-738Google Scholar). A cDNA clone from rat brain encoding an aminotransferase with KAT activity has been recently isolated(3Alberati-Giani D. Malherbe P. Köhler C. Lang G. Kiefer V. Cesura A.M. J. Neurochem. 1995; 64: 1448-1455Google Scholar), and it has been found that this protein corresponds to the rat kidney β-lyase/GTK enzyme cloned by Perry et al.(4Perry S.J. Schofield M.A. MacFarlaine M. Lock E.A. King L.J. Gibson G.G. Goldfarb P. Mol. Pharmacol. 1993; 43: 660-665Google Scholar). Rat brain also contains a mitochondrial form of this aminotransferase, which differs from cytosolic KAT/GTK exclusively for the presence of an NH2-terminal leader peptide, which targets the protein to the mitochondrial matrix(15Malherbe P. Alberati-Giani D. Köhler C. Cesura A.M. FEBS Lett. 1995; 367: 141-144Google Scholar). Whereas it has been claimed that in rats, a single pyruvate-preferring enzyme with KAT activity may be of physiological relevance in the cerebral synthesis of kynurenic acid(16Okuno E. Du F. Ishikawa T. Tsujimoto M. Nakamura M. Schwarcz R. Kido R. Brain Res. 1990; 534: 37-44Google Scholar, 17Okuno E. Schmidt W. Parks D.A. Nakamura M. Schwarcz R. J. Neurochem. 1991; 57: 533-540Google Scholar), whether in rat brain other aminotransferase forms are involved in the biosynthesis of kynurenic acid remains to be established. Noteworthy, the presence of AadAT activity has also been described in rat brain (see (18Huck S. Grass F. Hörtnagl H. J. Neurosci. 1984; 4: 2650-2657Google Scholar) and references therein), therefore suggesting that this aminotransferase may represent a second synthetic enzyme for kynurenic acid in the central nervous system of this animal species.In the present work, we describe the molecular cloning and the functional expression of a soluble aminotransferase from rat kidney displaying both KAT and AadAT activity (KAT/AadAT).