KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to α-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15–33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

INTRODUCTION

Aminotransferase, capable of catalysing the transamination of kynurenine to KYNA (kynurenic acid), has commonly been termed KAT (kynurenine aminotransferase). KYNA is the only known endogenous antagonist of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptors [14]. KYNA is also the antagonist of the α7-nicotinic acetylcholine receptor [58]. The level of KYNA is altered in several neurodegenerative diseases, including Huntington's disease [9,10], Alzheimer's disease [11], schizophrenia [1214] and acquired immunodeficiency syndrome dementia [15]. Because KYNA must be produced by KAT-catalysed kynurenine transamination, aminotransferases, responsible for catalysing kynurenine to KYNA, have been considered to be targets for maintaining and regulating physiological concentrations of brain KYNA.

In humans, rats and mice, four enzymes, KAT I, II, III and IV, are considered to be involved in KYNA synthesis in the central nervous system [1621]. Of these, KAT I and KAT II have been extensively studied and the crystal structures of hKAT I (human kynurenine aminotransferase I) and hKAT II (human kynurenine aminotransferase II) are now available [16,17,19,2225]. It has been reported that KAT I has a broad substrate specificity and displays maximum activity at relatively basic conditions. This poses critical questions regarding the contribution of KAT I to brain KYNA production [16,17]. On the other hand, KAT II is identical to AADAT (aminoadipate aminotransferase) and catalyses the transamination of kynurenine and aminoadipate and is localized in the soluble cytoplasm [16]. The enzyme has been cloned from both rats and humans and its presence in the brain has been confirmed by Northern and Western blotting [2629]. Previous studies on KAT II/AADAT have been focused on its activity on aminoadipate in peripheral organs, especially in the liver and the kidney, and on its role in brain KYNA production [3032].

Although there have been a number of studies addressing the biochemical characterization of KAT II/AADAT, the overall substrate specificity and kinetic properties have not been clearly established for KAT II from any species. In the present study, we determined its overall substrate profile, kinetic parameters and the complex structure of hKAT II with α-oxoglutaric acid. The present study shows a novel biochemical characterization of hKAT II.

MATERIALS AND METHODS

Expression, purification and activity assay of recombinant hKAT II

Protein expression and purification

Recombinant hKAT II coding sequence (GenBank® Nucleotide Sequence accession number NM_182662) was cloned into an Impact™-CN plasmid (New England Biolabs) for expression of a fusion protein containing a chitin-binding domain. hKAT II was purified by affinity purification, DEAE–Sepharose, hydroxyapatite and gel-filtration chromatography. The purified recombinant hKAT II was concentrated to 10 mg·ml−1 of protein in 10 mM phosphate buffer (pH 7.4) using a Centricon YM-30 concentrator (Millipore) [24].

Activity assay

The KAT activity assay was based upon methods described previously [33]. Briefly, a reaction mixture (50 μl final volume) containing 10 mM L-kynurenine, 2 mM α-oxoglutarate, 40 μM PLP (pyridoxal 5′-phosphate) and 2 μg of the protein sample was prepared in 100 mM potassium phosphate buffer (pH 7.4). The mixture was incubated at 37°C for 15 min, and the reaction was stopped by the addition of an equal volume of 0.8 M formic acid. The supernatant of the reaction mixture, obtained by centrifugation at 15000 g for 10 min at 4°C, was analysed by HPLC, with UV detection performed at 330 nm for both kynurenine and KYNA.

Effect of pH and temperature on hKAT II

To determine the effect of the buffer pH on hKAT II activity, a buffer mixture consisting of 100 mM phosphate and 100 mM boric acid was prepared, and the pH of the buffer was adjusted to pH 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0. The buffer mixture was selected to maintain a relatively consistent buffer composition and ionic strength, but would have sufficient buffering capacity for a relatively broad range of pH values. A typical reaction mixture containing 10 mM kynurenine, 2 mM α-oxoglutarate and 2 μg of hKAT II was prepared using the buffer mixture at different pHs. The reaction mixture was incubated and analysed in the hKAT II activity assay as described above. To determine the effect of temperature on hKAT II-catalysed kynurenine transamination, the typical reaction mixture [final volume of 50 μl in 100 mM phosphate buffer (pH 7.4)] was incubated at temperatures ranging from 10–80°C for 15 min and the product formed in the mixture was analysed as described above for the hKAT II activity assay.

Substrate specificity

To test the transamination activity of other amino acids, an assay mixture containing 15 mM amino acid, 20 mM glyoxylate or α-oxobutyrate (for glycine), 40 μM PLP, 100 mM potassium phosphate buffer (pH 7.4) and 2 μg of enzyme was prepared in a total volume of 50 μl. The mixture was incubated for 15 min at 37°C. The product was quantified based on the detection of the OPT (O-phthaldialdehyde thiol)–amino-acid product conjugate by HPLC, with electrochemical detection performed after their corresponding reaction mixtures were derivatized using an OPT reagent [34]. In a kinetic study for amino-acid substrates, the typical reaction mixture specified in the KAT activity assay [50 μl final volume prepared in 100 mM phosphate buffer (pH 7.4)] containing various concentrations (0.47–30 mM) of the amino acids which showed detectable transaminase activity with hKAT II and 30 mM glyoxylate was used. The mixture was incubated for 15 min at 37°C. To determine the substrate specificity for α-oxo acids, 16 individual α-oxo acids were tested for their ability to function as the amino-group acceptor for hKAT II. In the assays, the typical reaction mixture stated above also contained a different α-oxo acid at various concentrations (0.47–60 mM) and 15 mM kynurenine, and the rate of KYNA production was determined by performing the KAT activity assay as described above. The kinetic parameters were calculated by fitting the Michaelis–Menten equation to the experimental data using the enzyme kinetics module in SigmaPlot 8.2 (SPSS).

The effect of other amino acids on KAT enzyme activity

Analysis of the substrate specificity revealed that a number of amino acids could serve as the amino-group donor for hKAT II. To determine the effect of other amino acids on hKAT II-catalysed KYNA production, a different amino acid (final concentration of 5 mM) was incorporated into the typical reaction mixture (total volume of 50 μl) which contained 5 mM kynurenine, 20 mM glyoxylate and 2 μg of hKAT II (pH 7.4) and the enzyme activity was assayed in the same manner as described above for the KAT activity assay.

Crystal structure of the hKAT II complex with α-oxoglutaric acid

The crystals were grown using the hanging-drop vapour diffusion methods described previously [24]. The crystals of the enzyme–α-oxoglutaric acid complex were obtained by soaking the crystals in 2.5 mM α-oxoglutaric acid in the crystallization buffer for 3 days. Individual hKAT II complex crystals were cryogenized using 25% (v/v) glycerol in the crystallization buffer as a cryoprotectant solution. Diffraction data for the crystal were collected at the Brookhaven National Synchrotron Light Source beam line X29A [λ=1.0908 Å (1 Å=0.1 nm)]. All data were indexed and integrated using HKL-2000 software (HKL Research), and scaling and merging of the diffraction data were performed using the program SCALEPACK [35]. The parameters of the crystals and data collection are listed in Table 1. The structure of hKAT II was determined using the molecular-replacement method using the hKAT II structure (Protein Data Bank code 2QLR). The program Molrep [36] was employed to calculate both cross-rotation and translation functions of the model. The initial model was subjected to iterative cycles of crystallographic refinement with the Refmac 5.2 [37] and graphic sessions for model building using the program O [38]. Solvent molecules were automatically added and refined with ARP/wARP [39] together with Refmac 5.2. Superposition of the structures was done using Lsqkab [40] in the CCP4 suite, and Figures were generated using PyMOL [41].

Table 1
Data collection and refinement statistics of the hKAT II complex with α-oxoglutaric acid

GOL, glycerol; 2OG, α-oxooglutaric acid; RMS, root mean square.

Crystal data
 Space group P1 
 Unit cell  
 a (Â) 60.9 
 b (Â) 72.1 
 c (Â) 109.1 
 α (°) 90.0 
 β (°) 100.7 
 γ (°) 93.9 
Data collection  
 X-ray source Brookhaven National 
  Laboratory-X29 
 Wavelength (Â) 1.0809 
 Resolution (Â)* 2.48 (2.55–2.48) 
 Total number of reflections 193082 
 Number of unique reflections 63835 
R-merge* 0.11 (0.30) 
 Redundancy* 3.4 (2.7) 
 Completeness (%)* 89.6 (59.7) 
Refinement statistics  
R-work (%) 21.5 
R-free (%) 24.4 
 RMS bond lengths (Â) 0.020 
 RMS bond angles (°) 1.857 
 Number of ligand or cofactor molecules 4 LLP 
 1 2OG 
 7 GOL 
 Number of water molecules 828 
 Average B overall (Â233.9 
Crystal data
 Space group P1 
 Unit cell  
 a (Â) 60.9 
 b (Â) 72.1 
 c (Â) 109.1 
 α (°) 90.0 
 β (°) 100.7 
 γ (°) 93.9 
Data collection  
 X-ray source Brookhaven National 
  Laboratory-X29 
 Wavelength (Â) 1.0809 
 Resolution (Â)* 2.48 (2.55–2.48) 
 Total number of reflections 193082 
 Number of unique reflections 63835 
R-merge* 0.11 (0.30) 
 Redundancy* 3.4 (2.7) 
 Completeness (%)* 89.6 (59.7) 
Refinement statistics  
R-work (%) 21.5 
R-free (%) 24.4 
 RMS bond lengths (Â) 0.020 
 RMS bond angles (°) 1.857 
 Number of ligand or cofactor molecules 4 LLP 
 1 2OG 
 7 GOL 
 Number of water molecules 828 
 Average B overall (Â233.9 
*

The values in parentheses are for the highest resolution shell.

RESULTS

Biophysical properties of hKAT II

On the basis of SDS/PAGE analysis, very high purity of the isolated hKAT II was achieved. The isolated hKAT II was stable under freezing conditions (results not shown). No noticeable decrease in specific activity was observed when aliquots of hKAT II were stored at −80°C during a period of 6 months, which allowed us to analyse critically the substrate specificity and kinetic properties of hKAT II using aliquots of the enzyme from the same batch of highly purified hKAT II. The enzyme showed maximum activity at approx. 50°C and had a broad optimum pH range from pH 7–9 (Figure 1), which is similar to those reported previously [16,17].

Effect of pH and temperature on enzyme activity

Figure 1
Effect of pH and temperature on enzyme activity

The activities of recombinant hKAT II at different temperatures (A) and pH values (B). T, temperature. Results are means±S.D. (n=2).

Figure 1
Effect of pH and temperature on enzyme activity

The activities of recombinant hKAT II at different temperatures (A) and pH values (B). T, temperature. Results are means±S.D. (n=2).

Substrate study of hKAT II

hKAT II was tested for activity towards 24 amino acids and 16 oxo acids. Of the 24 amino acids tested, hKAT II was able to catalyse transamination of 20 of the amino acids. The specific activity of hKAT II towards individual amino acids under the conditions applied is shown in Figure 2. This substrate screening assay determined that hKAT II has a very broad substrate specificity. Kinetic analysis of hKAT II resulted in the determination of the catalytic efficiency of the enzyme towards individual amino-group donors and acceptors (Table 2), which provided a basis to determine its primary amino-group donors and acceptors and for speculation regarding its primary physiological functions. On the basis of the kinetic data, hKAT II should be equally efficient in catalysing the transamination of aminoadipate, kynurenine, methionine and glutamate (Table 2). All 16 α-oxo acids tested were capable of serving as amino-group acceptors. Of these, α-oxoglutarate, α-oxocaproic acid, phenylpyruvate and α-KMB (α-oxo-γ-methiol-butyric acid) were highly efficient as amino-group acceptors for hKAT II (Table 2). Although glyoxylate showed a relatively low affinity for hKAT II, it did not inhibit hKAT activity at high concentrations and its glycine–OPT complex was easily resolved from all other amino acid–OPT complexes during HPLC analysis with electrochemical detection. Therefore glyoxylate has been used as the primary amino-group acceptor for screening of the amino-group donors of hKAT II and kinetic analysis of the enzyme in the presence of different amino-group donors. KYNA displayed an absorbance peak with a λmax of approx. 330 nm and was easily resolved from kynurenine during reverse-phase separation; therefore kynurenine was used as the primary amino-group donor during screening of amino-group acceptors and their kinetic analysis.

Effect of other amino acids on hKAT II/KAT activity

Figure 2
Effect of other amino acids on hKAT II/KAT activity

The activities were quantified by measuing the amount of KYNA produced in the reaction mixtures. The rate of KYNA production in the hKAT II-containing reaction mixtures is shown. Results are means±S.D. (n=4). 3-HK, 3-hydroxy-kynurenine; GABA, γ-aminobutyric acid; Kyn, kynurenine.

Figure 2
Effect of other amino acids on hKAT II/KAT activity

The activities were quantified by measuing the amount of KYNA produced in the reaction mixtures. The rate of KYNA production in the hKAT II-containing reaction mixtures is shown. Results are means±S.D. (n=4). 3-HK, 3-hydroxy-kynurenine; GABA, γ-aminobutyric acid; Kyn, kynurenine.

Table 2
Kinetic parameters of hKAT II towards amino acids and α-oxo acids

The Km and kcat for oxo acids were derived by using various concentrations of individual oxo acids in the presence of 10 mM kynurenine; for amino acids, the results were derived by using various concentrations of individual amino acids in the presence of 30 mM glyoxylate. 3-HK, 3-HK, 3-hydroxy-kynurenine; α-KMB, α-oxo-γ-methiol-butyric acid. Results are means±S.E.M. (n=2).

Km (mM)kcat (min−1)kcat/Km (min−1·mM−1)
Amino-acid substrates    
 Aminoadipate 0.9±0.1 179.4±5.7 196.2 
 Kynurenine 4.7±0.8 585.3±39.9 125.9 
 Methionine 1.7±0.5 206.4±17 123.8 
 Glutamate 1.6±0.4 185.0±14.8 118.7 
 Tyrosine 1.8±0.2 131.5±5.2 74.4 
 Phenylalanine 5.2±1.7 327.1±46.2 63.4 
 Tryptophan 4.3±0.4 254.0±8.6 58.7 
 Leucine 5.1±3.0 285.0±70.4 56.1 
 3-HK 3.8±1.0 102.1±12.2 26.8 
 Glutamine 8.1±2.9 95.5±16.6 11.8 
 Alanine 19.4±9.2 173.6±43.2 9.0 
 Aminobutyrate 17.8±4.3 157.2±19.4 8.8 
Oxo acid substrates    
 α-Oxoglutarate 1.2±0.4 460.1±49.8 374.5 
 α-Oxocaproic acid 1.5±0.1 289.7±8.1 188.0 
 Phenylpyruvate 1.8±0.2 284.0±13 156.9 
 α-KMB 2.4±0.7 256.2±31.9 105.2 
 Mercaptopyruvate 2.8±0.5 215.5±12.6 77.7 
 Indo-3-pyruvate 1.4±1.2 89.1±32.5 64.6 
 α-Oxovalerate 3.4±0.3 159.3±4.9 47.0 
 α-Oxoleucine 3.3±0.8 150.5±13.6 45.4 
 α-Oxobutyrate 12.7±2.1 208.9±12.5 16.4 
 Hydroxyphenylpyruvate 1.5±1.1 23.6±9.5 16.2 
 α-Oxoadipate 20.9±5 290.3±30 13.9 
 Glyoxylate 18.0±3.7 218.4±18.4 12.1 
 Oxaloacetate 16.8±12.4 93.9±51.6 5.6 
 α-Oxovaline 12.9±2.9 55.1±4.6 4.3 
 α-Oxoisoleucine 14.2±3.8 55.2±6.9 3.9 
 Pyruvate 9.7±5.6 21.8±6.4 2.3 
Km (mM)kcat (min−1)kcat/Km (min−1·mM−1)
Amino-acid substrates    
 Aminoadipate 0.9±0.1 179.4±5.7 196.2 
 Kynurenine 4.7±0.8 585.3±39.9 125.9 
 Methionine 1.7±0.5 206.4±17 123.8 
 Glutamate 1.6±0.4 185.0±14.8 118.7 
 Tyrosine 1.8±0.2 131.5±5.2 74.4 
 Phenylalanine 5.2±1.7 327.1±46.2 63.4 
 Tryptophan 4.3±0.4 254.0±8.6 58.7 
 Leucine 5.1±3.0 285.0±70.4 56.1 
 3-HK 3.8±1.0 102.1±12.2 26.8 
 Glutamine 8.1±2.9 95.5±16.6 11.8 
 Alanine 19.4±9.2 173.6±43.2 9.0 
 Aminobutyrate 17.8±4.3 157.2±19.4 8.8 
Oxo acid substrates    
 α-Oxoglutarate 1.2±0.4 460.1±49.8 374.5 
 α-Oxocaproic acid 1.5±0.1 289.7±8.1 188.0 
 Phenylpyruvate 1.8±0.2 284.0±13 156.9 
 α-KMB 2.4±0.7 256.2±31.9 105.2 
 Mercaptopyruvate 2.8±0.5 215.5±12.6 77.7 
 Indo-3-pyruvate 1.4±1.2 89.1±32.5 64.6 
 α-Oxovalerate 3.4±0.3 159.3±4.9 47.0 
 α-Oxoleucine 3.3±0.8 150.5±13.6 45.4 
 α-Oxobutyrate 12.7±2.1 208.9±12.5 16.4 
 Hydroxyphenylpyruvate 1.5±1.1 23.6±9.5 16.2 
 α-Oxoadipate 20.9±5 290.3±30 13.9 
 Glyoxylate 18.0±3.7 218.4±18.4 12.1 
 Oxaloacetate 16.8±12.4 93.9±51.6 5.6 
 α-Oxovaline 12.9±2.9 55.1±4.6 4.3 
 α-Oxoisoleucine 14.2±3.8 55.2±6.9 3.9 
 Pyruvate 9.7±5.6 21.8±6.4 2.3 

Effects of other amino acids on hKAT II-catalysed kynurenine transamination

On the basis of the Km of hKAT II with different amino acids, it seems apparent that some amino-acid substrates are likely to inhibit hKAT II activity towards kynurenine. When each of the 23 amino acids (at 5 mM concentration) was incorporated with 5 mM kynurenine into the kynurenine/hKAT II/α-oxo acid reaction mixture, aminoadipate, asparagine, glutamate, histidine, cysteine, lysine, 3-hydroxy-kynurenine and phenylalanine were all shown to decrease the rate of KYNA formation, but none of them inhibited more than 35% of KAT activity (Figure 2).

Overall structure

The crystal structure of the hKAT II–α-oxoglutaric acid complex was determined by molecular replacement and refined to a resolution of 2.48 Å. The final model yields a crystallographic R value of 21.5% and an Rfree value of 24.4% with ideal geometry (Table 1). There are four protein molecules that form two biological homodimers in an asymmetric unit and the protein residues are numbered 1(A)–425(A) for molecule A, 1(B)–425(B) for molecule B, 1(C)–425(C) for molecule C, and 1(D)–425(D) for molecule D. Pro18 (A) to Gly29 (A), Arg20 (B) to Gly29 (B), and Arg20 (D) to Arg28 (D) have not been included in the final hKAT II–α-oxoglutaric acid complex model because they are highly disordered. To distinguish the residues from the two subunits of a biological dimer in the description of the substrate interaction, residues from the opposite subunit have been labelled with an asterisk (*). The results of the refinement are summarized in Table 1.

The structural basis of the broad substrate specificity of hKAT II

We were able to model α-oxoglutaric acid in one of the four subunits. In the other three subunits, the equivalent position is occupied by glycerol. Inspection of the active centre of the hKAT II crystal structure revealed that the substrate lies near the PLP molecule that forms LLP (lysine pyridoxal-5′-phosphate) with Lys263. Several residues, including Tyr142 and Asn202 from one subunit, and Gly39* and Tyr74* from the opposite subunit, define the substrate-binding site and contact the α-oxoglutaric acid molecule. The ring of Tyr74* has a weak hydrophobic interaction with α-oxoglutaric acid, and its hydroxy group forms a weak hydrogen bond with the O2 atom of α-oxoglutaric acid (Figure 3). The interactions of glycerol and the hKAT II active-centre residues include the formation of hydrogen bonds between glycerol and residues Tyr142, Gln118 and Tyr233 and hydrophobic interactions between glycerol and residues Tyr74* and Leu19* from the opposite subunit (Figure 4). Upon superposing of the hKAT II subunit B (with α-oxoglutaric acid) and hKAT II subunit D (with glycerol) on to the hKAT II–kynurenine structure (Figure 5), we identified the residues within 5 Å of the ligands. α-Oxoglutaric acid, glycerol and kynurenine basically occupy similar positions, but α-oxoglutaric acid has fewer interactions with the enzyme than kynurenine. The carboxylic group of the α-oxoglutaric acid substrate does not form salt bridges with the guanidinium group of Arg399. PLP forms LLP with Lys263 when the subunit binds glycerol or α-oxoglutaric acid, and forms PMP (pyridoximine 5′-phosphate) when the protein binds kynurenine. Most residues within 5 Å have the same conformation, except for a few residues in the N-terminal fragment (residues 15–33) (Figure 5). When the protein molecule binds glycerol, this N-terminal fragment moves towards the centre to interact with glycerol. However, when it binds kynurenine, the N-terminal fragment moves away from the centre and leaves room for kynurenine. Also, when it binds α-oxoglutaric acid, the N-terminal fragment seems to move further away to leave more space for the substrate. In summary, the finding that the N-terminal fragment is disordered simply indicates that the N-terminal fragment is highly flexible and is moved upon binding of the substrate.

α-Oxoglutaric acid binding site

Figure 3
α-Oxoglutaric acid binding site

α-Oxoglutaric acid (2OG) and the protein residues within a distance of 4 Â of 2OG are shown (distances are stated in Â). The 2Fo-Fc electron-density map covering 2OG is contoured at the 1.1σ level. The Figure was generated by PyMOL. * indicates residues on the opposite subunit.

Figure 3
α-Oxoglutaric acid binding site

α-Oxoglutaric acid (2OG) and the protein residues within a distance of 4 Â of 2OG are shown (distances are stated in Â). The 2Fo-Fc electron-density map covering 2OG is contoured at the 1.1σ level. The Figure was generated by PyMOL. * indicates residues on the opposite subunit.

Glycerol-binding site in the active centre

Figure 4
Glycerol-binding site in the active centre

Glycerol (GOL) and the protein residues within a 4 Â distance of GOL are shown (distances are stated in Â). The 2Fo-Fc electron-density map covering GOL is contoured at the 1.1σ level. The Figure was generated by PyMOL. * indicates residues on the opposite subunit.

Figure 4
Glycerol-binding site in the active centre

Glycerol (GOL) and the protein residues within a 4 Â distance of GOL are shown (distances are stated in Â). The 2Fo-Fc electron-density map covering GOL is contoured at the 1.1σ level. The Figure was generated by PyMOL. * indicates residues on the opposite subunit.

Superposition of the hKAT II subunit B (with α-oxoglutaric acid, green) and hKAT II subunit D (with glycerol, yellow) on to the hKAT II–kynurenine structure (pink)

Figure 5
Superposition of the hKAT II subunit B (with α-oxoglutaric acid, green) and hKAT II subunit D (with glycerol, yellow) on to the hKAT II–kynurenine structure (pink)

The major protein conformation changes involved in ligand binding are shown in ribbon form (residues 15–33). GOL, glycerol; Kyn, kynurenine; LLP263, PLP formed with Lys263; 2OG, α-oxoglutaric acid, PMP, pyridoximine 5′-phosphate.

Figure 5
Superposition of the hKAT II subunit B (with α-oxoglutaric acid, green) and hKAT II subunit D (with glycerol, yellow) on to the hKAT II–kynurenine structure (pink)

The major protein conformation changes involved in ligand binding are shown in ribbon form (residues 15–33). GOL, glycerol; Kyn, kynurenine; LLP263, PLP formed with Lys263; 2OG, α-oxoglutaric acid, PMP, pyridoximine 5′-phosphate.

DISCUSSION

AADAT was first detected in rat liver [42] and its potential function in liver lysine degradation has been well described [43]. It was reported that the same aminotransferase from liver and kidney had both KAT activity and AADAT activity [4446] and the true identity of rat KAT II and AADAT was confirmed by Tobes and Mason [45].

KAT II has been considered to be the principal enzyme responsible for the synthesis of KYNA in the rodent and human brain [17,18]. Mice in which KAT II was knocked out by genomic manipulation showed a substantial decrease in brain KYNA levels and displayed phenotypic changes that were in line with this chemical deficit [7,47,48]. In the present study, kinetic analysis shows a better overall catalytic efficiency of hKAT II towards kynurenine than towards other amino-group donors, except for aminoadipate. Although hKAT II has a relatively high Km for kynurenine, its catalytic efficiency in the kynurenine to KYNA pathway is enhanced by its relatively high catalytic-centre activity towards kynurenine and also by the irreversible nature of the kynurenine to KYNA pathway. The enzymatic product of kynurenine transamination is an α-oxo acid intermediate that is unstable and undergoes rapid intramolecular cyclization to KYNA. Consequently, no direct enzymatic product accumulates during KAT-catalysed kynurenine transamination and the equilibrium always proceeds towards KYNA production. Therefore the transamination of kynurenine to KYNA is one of the major functions of hKAT II.

Although most of the previous studies on KAT II have emphasized its function in KYNA biosynthesis because of the important role of KYNA in the central nervous system, the enzyme has a higher catalytic efficiency for aminoadipate than for kynurenine (Table 2). Human liver has been shown to have AADAT activity [49]. In rats, AADAT activity has been found in liver and kidney tissues [42,4446,5052]. In humans, lysine is the main source of aminoadipate. Lysine, an essential amino acid in humans, is degraded when there is an excess present beyond that required for protein synthesis. The aminoadipate pathway is the major route for the catabolism of lysine and occurs in the liver [27,53,54]. From the enzyme kinetic parameters in this paper, we showed that aminoadipate was the best amino-acid substrate for hKAT II. The fact that α-oxoadipate is not a good amino-group acceptor for the enzyme suggests that the AADAT enzyme reaction does not tend to be reversible, and favours the direction from aminoadipate to α-oxoadipate, one of the reactions involved in lysine degradation (Figure 6). An additional argument for a primary role of hKAT II in the aminoadipate to α-oxoadipate pathway comes from the comparison of hKAT II with its homologues from some micro-organisms. The KAT II or AADAT present in lower eukaryotes and certain bacteria is involved primarily in lysine biosynthesis (the reversal of lysine catabolism) because it catalyses the transamination reaction using α-oxoadipate as an amino acceptor to yield aminoadipate [5558]. The kinetic constant (Km) of a yeast KAT II homologue is 14 mM for α-oxoadipate and 20 mM for aminoadipate [59]. The homologue enzyme from Thermus thermophilus catalyses efficiently the transamination from α-oxoadipate to aminoadipate with negligible activity in the reverse reaction [55]. These results show that the KAT II homologues in yeast and T. thermophilus favour the transamination direction from α-oxoadipate to aminoadipate, the reaction involved in lysine biosynthesis. In contrast, the affinity of hKAT II for aminoadipate (Km=0.9 mM) is much greater than its affinity for oxoadipate (Km=20.9 mM), which indirectly supports a role for hKAT II in lysine degradation.

Lysine catabolism pathway in mammals

Figure 6
Lysine catabolism pathway in mammals
Figure 6
Lysine catabolism pathway in mammals

Aminoadipate occurs naturally in the brain [53,60]. The compound is known to be a selective toxin for astrocytes and related glial cells because injection of a racemic mixture of aminoadipate induced transient degenerative changes in non-neuronal cells [61]. Its toxicity to glial cells has been further demonstrated by a number of subsequent in vivo or in vitro studies [6269]. Since KAT II is present in the astrocyte-like cells of mammalian brains [28,70,71], it raises a reasonable argument for the role of KAT II in the neutralization of aminoadipate in the mammalian brain. On the other hand, many previous studies have shown that a high concentration of aminoadipate inhibits glutamate transport [72,73], blocks glutamine synthetase [74], prevents the uptake of glutamate into synaptic vesicles [75] and functions as an NMDA receptor agonist [76]. These effects can contribute to an increased excitatory tone because synaptic glutamate concentrations are elevated. Therefore the hyperactive behaviour observed in katII−/− mice [47] raised an interesting question as to whether the aminoadipate level is increased in the knockout mouse brain and consequently contributes to the pathological mechanisms of the abnormal behaviour. High levels of aminoadipate in serum and/or urine have also been observed in many cases with neurological and other disorders [7787].

Biochemical analysis indicates that hKAT II is a multifunctional aminotransferase. Kinetic analysis of the enzyme towards different amino acids and oxo acids showed that the enzyme was efficient in catalysing the transamination of a number of other amino acids, as well as kynurenine and α-aminoadipate, and could use many α-oxo acids as amino-group acceptors. The characterization of multiple substrates for hKAT II suggests that the enzyme might play a role in the metabolism of other oxo acids and amino acids, which requires further study. It also raises a fundamental question regarding the structural basis of its broad substrate specificity. A previous study [24] showed that a major conformational change of residues 16–31 in hKAT II is involved in substrate binding and for shielding the substrate-binding pocket from bulk solvents. The hKAT II–α-oxoglutaric acid structure shows that this N-terminal fraction is very flexible and it has different conformations when the protein subunit binds to different ligands. This N-terminal fraction moves close to the active centre when the protein subunit binds a small ligand, and moves away from the centre when the subunit binds a large ligand. In the hKAT II–α-oxoaglutarate structure we did not find any other large-scale conformational changes, such as domain–domain rotation, which is involved in binding substrates in other aminotransferases [8894]. So the conformational change of part of the N-terminal residues is the main mechanism which adapts to accommodate various substrates of different sizes in the active centre.

In summary, there have been a number of previous studies [16,17,4446] dealing with the biochemical characterization of KAT II/AADAT, which emphasize either the KAT activity of the enzyme or its AADAT function. The present study, however, produces an overall picture regarding the substrate profiles of hKAT II, its catalytic efficiency for different amino-group donors and acceptors and its structural basis of substrate binding and catalysis. Our results suggest that hKAT II is likely to play primary roles in both the kynurenine to KYNA and aminoadipate to α-oxoadipate pathways. In addition, the enzyme may be involved in the regulation of other amino acids and α-oxo acids. Therefore it will be interesting to study the overall functions of hKAT II in vivo in the future, particularly in order to use the available Kat II gene-deficient mice to test any possible hypotheses.

Abbreviations

     
  • AADAT

    aminoadipate aminotransferase

  •  
  • KAT

    kynurenine aminotransferase

  •  
  • hKAT II

    human kynurenine aminotransferase II

  •  
  • KYNA

    kynurenic acid

  •  
  • LLP

    lysine pyridoxal-5′-phosphate

  •  
  • NMDA

    N-methyl-D-aspartate

  •  
  • OPT

    O-phthaldialdehyde thiol

  •  
  • PLP

    pyridoxal 5′-phosphate

This work was carried out in part at the National Synchrotron Light Source, Brookhaven National Laboratory (Upton, NY 11973, U.S.A.). This research was supported in part by the Intramural Research Program of the institutes of the NIDCR and NINDS at the NIH (National Institutes of Health).

References

References
Leeson
 
P. D.
Iversen
 
L. L.
 
The glycine site on the NMDA receptor: structure–activity relationships and therapeutic potential
J. Med. Chem.
1994
, vol. 
37
 (pg. 
4053
-
4067
)
Perkins
 
M. N.
Stone
 
T. W.
 
An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid
Brain Res.
1982
, vol. 
247
 (pg. 
184
-
187
)
Stone
 
T. W.
Perkins
 
M. N.
 
Actions of excitatory amino acids and kynurenic acid in the primate hippocampus: a preliminary study
Neurosci. Lett.
1984
, vol. 
52
 (pg. 
335
-
340
)
Birch
 
P. J.
Grossman
 
C. J.
Hayes
 
A. G.
 
Kynurenic acid antagonises responses to NMDA via an action at the strychnine-insensitive glycine receptor
Eur. J. Pharmacol.
1988
, vol. 
154
 (pg. 
85
-
87
)
Pereira
 
E. F.
Hilmas
 
C.
Santos
 
M. D.
Alkondon
 
M.
Maelicke
 
A.
Albuquerque
 
E. X.
 
Unconventional ligands and modulators of nicotinic receptors
J. Neurobiol.
2002
, vol. 
53
 (pg. 
479
-
500
)
Hilmas
 
C.
Pereira
 
E. F.
Alkondon
 
M.
Rassoulpour
 
A.
Schwarcz
 
R.
Albuquerque
 
E. X.
 
The brain metabolite kynurenic acid inhibits α7 nicotinic receptor activity and increases non-α7 nicotinic receptor expression: physiopathological implications
J. Neurosci.
2001
, vol. 
21
 (pg. 
7463
-
7473
)
Alkondon
 
M.
Pereira
 
E. F.
Yu
 
P.
Arruda
 
E. Z.
Almeida
 
L. E.
Guidetti
 
P.
Fawcett
 
W. P.
Sapko
 
M. T.
Randall
 
W. R.
Schwarcz
 
R.
, et al 
Targeted deletion of the kynurenine aminotransferase ii gene reveals a critical role of endogenous kynurenic acid in the regulation of synaptic transmission via α7 nicotinic receptors in the hippocampus
J. Neurosci.
2004
, vol. 
24
 (pg. 
4635
-
4648
)
Stone
 
T. W.
 
Kynurenic acid blocks nicotinic synaptic transmission to hippocampal interneurons in young rats
Eur. J. Neurosci.
2007
, vol. 
25
 (pg. 
2656
-
2665
)
Beal
 
M. F.
Matson
 
W. R.
Swartz
 
K. J.
Gamache
 
P. H.
Bird
 
E. D.
 
Kynurenine pathway measurements in Huntington's disease striatum: evidence for reduced formation of kynurenic acid
J. Neurochem.
1990
, vol. 
55
 (pg. 
1327
-
1339
)
Guidetti
 
P.
Reddy
 
P. H.
Tagle
 
D. A.
Schwarcz
 
R.
 
Early kynurenergic impairment in Huntington's disease and in a transgenic animal model
Neurosci. Lett.
2000
, vol. 
283
 (pg. 
233
-
235
)
Widner
 
B.
Leblhuber
 
F.
Walli
 
J.
Tilz
 
G. P.
Demel
 
U.
Fuchs
 
D.
 
Tryptophan degradation and immune activation in Alzheimer's disease
J. Neural. Transm.
2000
, vol. 
107
 (pg. 
343
-
353
)
Schwarcz
 
R.
Rassoulpour
 
A.
Wu
 
H. Q.
Medoff
 
D.
Tamminga
 
C. A.
Roberts
 
R. C.
 
Increased cortical kynurenate content in schizophrenia
Biol. Psychiatry.
2001
, vol. 
50
 (pg. 
521
-
530
)
Erhardt
 
S.
Blennow
 
K.
Nordin
 
C.
Skogh
 
E.
Lindstrom
 
L. H.
Engberg
 
G.
 
Kynurenic acid levels are elevated in the cerebrospinal fluid of patients with schizophrenia
Neurosci. Lett.
2001
, vol. 
313
 (pg. 
96
-
98
)
Erhardt
 
S.
Schwieler
 
L.
Nilsson
 
L.
Linderholm
 
K.
Engberg
 
G.
 
The kynurenic acid hypothesis of schizophrenia
Physiol. Behav.
2007
, vol. 
92
 (pg. 
203
-
209
)
Guillemin
 
G. J.
Kerr
 
S. J.
Brew
 
B. J.
 
Involvement of quinolinic acid in AIDS dementia complex
Neurotox. Res.
2005
, vol. 
7
 (pg. 
103
-
123
)
Okuno
 
E.
Nakamura
 
M.
Schwarcz
 
R.
 
Two kynurenine aminotransferases in human brain
Brain Res.
1991
, vol. 
542
 (pg. 
307
-
312
)
Guidetti
 
P.
Okuno
 
E.
Schwarcz
 
R.
 
Characterization of rat brain kynurenine aminotransferases I and II
J. Neurosci. Res.
1997
, vol. 
50
 (pg. 
457
-
465
)
Schwarcz
 
R.
Pellicciari
 
R.
 
Manipulation of brain kynurenines: glial targets, neuronal effects, and clinical opportunities
J. Pharmacol. Exp. Ther.
2002
, vol. 
303
 (pg. 
1
-
10
)
Han
 
Q.
Li
 
J.
Li
 
J.
 
pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I
Eur. J. Biochem.
2004
, vol. 
271
 (pg. 
4804
-
4814
)
Yu
 
P.
Li
 
Z.
Zhang
 
L.
Tagle
 
D. A.
Cai
 
T.
 
Characterization of kynurenine aminotransferase III, a novel member of a phylogenetically conserved KAT family
Gene
2006
, vol. 
365
 (pg. 
111
-
118
)
Guidetti
 
P.
Amori
 
L.
Sapko
 
M. T.
Okuno
 
E.
Schwarcz
 
R.
 
Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain
J. Neurochem.
2007
, vol. 
102
 (pg. 
103
-
111
)
Rossi
 
F.
Han
 
Q.
Li
 
J.
Li
 
J.
Rizzi
 
M.
 
Crystal structure of human kynurenine aminotransferase I
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
50214
-
50220
)
Cooper
 
A. J.
Pinto
 
J. T.
Krasnikov
 
B. F.
Niatsetskaya
 
Z. V.
Han
 
Q.
Li
 
J.
Vauzour
 
D.
Spencer
 
J. P.
 
Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate β-lyase
Arch. Biochem. Biophys.
2008
, vol. 
474
 (pg. 
72
-
81
)
Han
 
Q.
Robinson
 
H.
Li
 
J.
 
Crystal structure of human kynurenine aminotransferase II
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
3567
-
3573
)
Rossi
 
F.
Garavaglia
 
S.
Montalbano
 
V.
Walsh
 
M. A.
Rizzi
 
M.
 
Crystal structure of human kynurenine aminotransferase II, a drug target for the treatment of schizophrenia
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
3559
-
3566
)
Buchli
 
R.
Alberati-Giani
 
D.
Malherbe
 
P.
Kohler
 
C.
Broger
 
C.
Cesura
 
A. M.
 
Cloning and functional expression of a soluble form of kynurenine/α-aminoadipate aminotransferase from rat kidney
J. Biol. Chem.
1995
, vol. 
270
 (pg. 
29330
-
29335
)
Goh
 
D. L.
Patel
 
A.
Thomas
 
G. H.
Salomons
 
G. S.
Schor
 
D. S.
Jakobs
 
C.
Geraghty
 
M. T.
 
Characterization of the human gene encoding α-aminoadipate aminotransferase (AADAT)
Mol. Genet. Metab.
2002
, vol. 
76
 (pg. 
172
-
180
)
Gramsbergen
 
J. B.
Schmidt
 
W.
Turski
 
W. A.
Schwarcz
 
R.
 
Age-related changes in kynurenic acid production in rat brain
Brain Res.
1992
, vol. 
588
 (pg. 
1
-
5
)
Yu
 
P.
Mosbrook
 
D. M.
Tagle
 
D. A.
 
Genomic organization and expression analysis of mouse kynurenine aminotransferase II, a possible factor in the pathophysiology of Huntington's disease
Mamm. Genome
1999
, vol. 
10
 (pg. 
845
-
852
)
Schwarcz
 
R.
 
The kynurenine pathway of tryptophan degradation as a drug target
Curr. Opin. Pharmacol.
2004
, vol. 
4
 (pg. 
12
-
17
)
Moroni
 
F.
 
Tryptophan metabolism and brain function: focus on kynurenine and other indole metabolites
Eur. J. Pharmacol.
1999
, vol. 
375
 (pg. 
87
-
100
)
Stone
 
T. W.
 
Neuropharmacology of quinolinic and kynurenic acids
Pharmacol. Rev.
1993
, vol. 
45
 (pg. 
309
-
379
)
Han
 
Q.
Fang
 
J.
Li
 
J.
 
3-Hydroxykynurenine transaminase identity with alanine glyoxylate transaminase. A probable detoxification protein in Aedes aegypti
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
15781
-
15787
)
Han
 
Q.
Fang
 
J.
Li
 
J.
 
Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase
Biochem. J.
2001
, vol. 
360
 (pg. 
617
-
623
)
Otwinowski
 
Z.
Minor
 
W.
 
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
1997
, vol. 
276
 (pg. 
307
-
326
)
Vagin
 
A.
Teplyakov
 
A.
 
MOLREP: an automated program for molecular replacement
J. Appl. Cryst.
1997
, vol. 
30
 (pg. 
1022
-
1025
)
Murshudov
 
G. N.
Vagin
 
A. A.
Dodson
 
E. J.
 
Refinement of macromolecular structures by the maximum-likelihood method
Acta Crystallogr., Sect. D: Biol. Crystallogr.
1997
, vol. 
53
 (pg. 
240
-
255
)
Jones
 
T. A.
Zou
 
J. Y.
Cowan
 
S. W.
Kjeldgaard
 
M.
 
Improved methods for building protein models in electron density maps and the location of errors in these models
Acta Crystallogr., Sect. A: Found. Crystallogr.
1991
, vol. 
47
 (pg. 
110
-
119
)
Perrakis
 
A.
Sixma
 
T. K.
Wilson
 
K. S.
Lamzin
 
V. S.
 
wARP: improvement and extension of crystallographic phases by weighted averaging of multiple refined dummy atomic models
Acta Crystallogr., Sect. D: Biol. Crystallogr.
1997
, vol. 
53
 (pg. 
448
-
455
)
Kabsch
 
W.
 
Crystal physics, diffraction, theoretical and general crystallography
Acta Crystallogr., Sect. A: Found. Crystallogr.
1976
, vol. 
32
 (pg. 
922
-
923
)
DeLano
 
W. L.
 
The PyMOL Molecular Graphics System
2002
CA, U.S.A.
Delano Scientifc, San Carlos
Nakatani
 
Y.
Fujioka
 
M.
Higashino
 
K.
 
α-Aminoadipate aminotransferase of rat liver mitochondria
Biochim. Biophys. Acta
1970
, vol. 
198
 (pg. 
219
-
228
)
Higashino
 
K.
Fujioka
 
M.
Yamamura
 
Y.
 
The conversion of L-lysine to saccharopine and α-aminoadipate in mouse
Arch. Biochem. Biophys.
1971
, vol. 
142
 (pg. 
606
-
614
)
Tobes
 
M. C.
Mason
 
M.
 
L-Kynurenine aminotransferase and L-α-aminoadipate aminotransferase. I. Evidence for identity
Biochem. Biophys. Res. Commun.
1975
, vol. 
62
 (pg. 
390
-
397
)
Tobes
 
M. C.
Mason
 
M.
 
α-Aminoadipate aminotransferase and kynurenine aminotransferase. Purification, characterization, and further evidence for identity
J. Biol. Chem.
1977
, vol. 
252
 (pg. 
4591
-
4599
)
Takeuchi
 
F.
Otsuka
 
H.
Shibata
 
Y.
 
Purification, characterization and identification of rat liver mitochondrial kynurenine aminotransferase with α-aminoadipate aminotransferase
Biochim. Biophys. Acta
1983
, vol. 
743
 (pg. 
323
-
330
)
Yu
 
P.
Di Prospero
 
N. A.
Sapko
 
M. T.
Cai
 
T.
Chen
 
A.
Melendez-Ferro
 
M.
Du
 
F.
Whetsell
 
W. O.
Guidetti
 
P.
Schwarcz
 
R.
Tagle
 
D. A.
 
Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice
Mol. Cell. Biol.
2004
, vol. 
24
 (pg. 
6919
-
6930
)
Sapko
 
M. T.
Guidetti
 
P.
Yu
 
P.
Tagle
 
D. A.
Pellicciari
 
R.
Schwarcz
 
R.
 
Endogenous kynurenate controls the vulnerability of striatal neurons to quinolinate: Implications for Huntington's disease
Exp. Neurol.
2006
, vol. 
197
 (pg. 
31
-
40
)
Okuno
 
E.
Tsujimoto
 
M.
Nakamura
 
M.
Kido
 
R.
 
2-Aminoadipate-2-oxoglutarate aminotransferase isoenzymes in human liver: a plausible physiological role in lysine and tryptophan metabolism
Enzyme Protein
1993
, vol. 
47
 (pg. 
136
-
148
)
Mawal
 
M. R.
Deshmukh
 
D. R.
 
α-Aminoadipate and kynurenine aminotransferase activities from rat kidney. Evidence for separate identity
J. Biol. Chem.
1991
, vol. 
266
 (pg. 
2573
-
2575
)
Mawal
 
M. R.
Deshmukh
 
D. R.
 
Purification and properties of α-aminoadipate aminotransferase from rat kidney
Prep. Biochem.
1991
, vol. 
21
 (pg. 
63
-
73
)
Mawal
 
M. R.
Mukhopadhyay
 
A.
Deshmukh
 
D. R.
 
Purification and properties of kynurenine aminotransferase from rat kidney
Biochem. J.
1991
, vol. 
279
 (pg. 
595
-
599
)
Chang
 
Y. E.
 
Lysine metabolism in the rat brain: the pipecolic acid-forming pathway
J. Neurochem.
1978
, vol. 
30
 (pg. 
347
-
354
)
Giacobini
 
E.
Nomura
 
Y.
Schmidt-Glenewinkel
 
T.
 
Pipecolic acid: origin, biosynthesis and metabolism in the brain
Cell. Mol. Biol. Incl. Cyto. Enzymol.
1980
, vol. 
26
 (pg. 
135
-
146
)
Miyazaki
 
T.
Miyazaki
 
J.
Yamane
 
H.
Nishiyama
 
M.
 
α-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus
Microbiology
2004
, vol. 
150
 (pg. 
2327
-
2334
)
Xu
 
H.
Andi
 
B.
Qian
 
J.
West
 
A. H.
Cook
 
P. F.
 
The α-aminoadipate pathway for lysine biosynthesis in fungi
Cell Biochem. Biophys.
2006
, vol. 
46
 (pg. 
43
-
64
)
Zabriskie
 
T. M.
Jackson
 
M. D.
 
Lysine biosynthesis and metabolism in fungi
Nat. Prod. Rep.
2000
, vol. 
17
 (pg. 
85
-
97
)
Matsuda
 
M.
Ogur
 
M.
 
Separation and specificity of the yeast glutamate-α-ketoadipate transaminase
J. Biol. Chem.
1969
, vol. 
244
 (pg. 
3352
-
3358
)
Matsuda
 
M.
Ogur
 
M.
 
Enzymatic and physiological properties of the yeast glutamate-α-ketoadipate transaminase
J. Biol. Chem.
1969
, vol. 
244
 (pg. 
5153
-
5158
)
Chang
 
Y. F.
 
Lysine metabolism in the human and the monkey: demonstration of pipecolic acid formation in the brain and other organs
Neurochem. Res.
1982
, vol. 
7
 (pg. 
577
-
588
)
Olney
 
J. W.
Ho
 
O. L.
Rhee
 
V.
 
Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system
Exp. Brain Res.
1971
, vol. 
14
 (pg. 
61
-
76
)
Olney
 
J. W.
de Gubareff
 
T.
Collins
 
J. F.
 
Stereospecificity of the gliotoxic and anti-neurotoxic actions of α-aminoadipate
Neurosci. Lett.
1980
, vol. 
19
 (pg. 
277
-
282
)
McBean
 
G. J.
 
Intrastriatal injection of DL-α-aminoadipate reduces kainate toxicity in vitro
Neuroscience
1990
, vol. 
34
 (pg. 
225
-
234
)
Huck
 
S.
Grass
 
F.
Hatten
 
M. E.
 
Gliotoxic effects of α-aminoadipic acid on monolayer cultures of dissociated postnatal mouse cerebellum
Neuroscience
1984
, vol. 
12
 (pg. 
783
-
791
)
Takada
 
M.
Hattori
 
T.
 
Fine structural changes in the rat brain after local injections of gliotoxin, α-aminoadipic acid
Histol. Histopathol.
1986
, vol. 
1
 (pg. 
271
-
275
)
Khurgel
 
M.
Koo
 
A. C.
Ivy
 
G. O.
 
Selective ablation of astrocytes by intracerebral injections of α-aminoadipate
Glia
1996
, vol. 
16
 (pg. 
351
-
358
)
Brown
 
D. R.
Kretzschmar
 
H. A.
 
The glio-toxic mechanism of α-aminoadipic acid on cultured astrocytes
J. Neurocytol.
1998
, vol. 
27
 (pg. 
109
-
118
)
Bridges
 
R. J.
Hatalski
 
C. G.
Shim
 
S. N.
Cummings
 
B. J.
Vijayan
 
V.
Kundi
 
A.
Cotman
 
C. W.
 
Gliotoxic actions of excitatory amino acids
Neuropharmacology
1992
, vol. 
31
 (pg. 
899
-
907
)
Nishimura
 
R. N.
Santos
 
D.
Fu
 
S. T.
Dwyer
 
B. E.
 
Induction of cell death by L-α-aminoadipic acid exposure in cultured rat astrocytes: relationship to protein synthesis
Neurotoxicology
2000
, vol. 
21
 (pg. 
313
-
320
)
Guidetti
 
P.
Hoffman
 
G. E.
Melendez-Ferro
 
M.
Albuquerque
 
E. X.
Schwarcz
 
R.
 
Astrocytic localization of kynurenine aminotransferase II in the rat brain visualized by immunocytochemistry
Glia
2007
, vol. 
55
 (pg. 
78
-
92
)
Kiss
 
C.
Ceresoli-Borroni
 
G.
Guidetti
 
P.
Zielke
 
C. L.
Zielke
 
H. R.
Schwarcz
 
R.
 
Kynurenate production by cultured human astrocytes
J. Neural Transm.
2003
, vol. 
110
 (pg. 
1
-
14
)
Pannicke
 
T.
Stabel
 
J.
Heinemann
 
U.
Reichelt
 
W.
 
α-Aminoadipic acid blocks the Na+-dependent glutamate transport into acutely isolated Muller glial cells from guinea pig retina
Pflugers Arch.
1994
, vol. 
429
 (pg. 
140
-
142
)
Schousboe
 
A.
Svenneby
 
G.
Hertz
 
L.
 
Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres
J. Neurochem.
1977
, vol. 
29
 (pg. 
999
-
1005
)
McBean
 
G. J.
 
Inhibition of the glutamate transporter and glial enzymes in rat striatum by the gliotoxin, α aminoadipate
Br. J. Pharmacol.
1994
, vol. 
113
 (pg. 
536
-
540
)
Fykse
 
E. M.
Iversen
 
E. G.
Fonnum
 
F.
 
Inhibition of L-glutamate uptake into synaptic vesicles
Neurosci. Lett.
1992
, vol. 
135
 (pg. 
125
-
128
)
Hall
 
J. G.
Hicks
 
T. P.
McLennan
 
H.
Richardson
 
T. L.
Wheal
 
H. V.
 
The excitation of mammalian central neurones by amino acids
J. Physiol.
1979
, vol. 
286
 (pg. 
29
-
39
)
Gray
 
R. G.
O'Neill
 
E. M.
Pollitt
 
R. J.
 
α-Aminoadipic aciduria: chemical and enzymatic studies
J. Inherit. Metab. Dis.
1980
, vol. 
2
 (pg. 
89
-
92
)
Fischer
 
M. H.
Brown
 
R. R.
 
Tryptophan and lysine metabolism in α-aminoadipic aciduria
Am. J. Med. Genet.
1980
, vol. 
5
 (pg. 
35
-
41
)
Casey
 
R. E.
Zaleski
 
W. A.
Philp
 
M.
Mendelson
 
I. S.
MacKenzie
 
S. L.
 
Biochemical and clinical studies of a new case of α-aminoadipic aciduria
J. Inherit. Metab. Dis.
1978
, vol. 
1
 (pg. 
129
-
135
)
Lee
 
J. S.
Yoon
 
H. R.
Coe
 
C. J.
 
A Korean girl with α-aminoadipic and α-ketoadipic aciduria accompanied with elevation of 2-hydroxyglutarate and glutarate
J. Inherit. Metab. Dis.
2001
, vol. 
24
 (pg. 
509
-
510
)
Sela
 
B. A.
Massos
 
T.
Fogel
 
D.
Zlotnik
 
J.
 
α-Amino adipic aciduria: a rare psychomotor syndrome
Harefuah
1999
, vol. 
137
 (pg. 
451
-
453
511
Candito
 
M.
Richelme
 
C.
Parvy
 
P.
Dageville
 
C.
Appert
 
A.
Bekri
 
S.
Rabier
 
D.
Chambon
 
P.
Mariani
 
R.
Kamoun
 
P.
 
Abnormal α-aminoadipic acid excretion in a newborn with a defect in platelet aggregation and antenatal cerebral haemorrhage
J. Inherit. Metab. Dis.
1995
, vol. 
18
 (pg. 
56
-
60
)
Takechi
 
T.
Okada
 
T.
Wakiguchi
 
H.
Morita
 
H.
Kurashige
 
T.
Sugahara
 
K.
Kodama
 
H.
 
Identification of N-acetyl-α-aminoadipic acid in the urine of a patient with α-aminoadipic and α-ketoadipic aciduria
J. Inherit. Metab. Dis.
1993
, vol. 
16
 (pg. 
119
-
126
)
Jakobs
 
C.
de Grauw
 
A. J.
 
A fatal case of 2-keto-, 2-hydroxy- and 2-aminoadipic aciduria: relation of organic aciduria to phenotype?
J. Inherit. Metab. Dis.
1992
, vol. 
15
 (pg. 
279
-
280
)
Vianey-Liaud
 
C.
Divry
 
P.
Cotte
 
J.
Teyssier
 
G.
 
α-Aminoadipic and α-ketoadipic aciduria: detection of a new case by a screening program using two-dimensional thin layer chromatography of amino acids
J. Inherit. Metab. Dis.
1985
, vol. 
8
 
Suppl. 2
(pg. 
133
-
134
)
Duran
 
M.
Beemer
 
F. A.
Wadman
 
S. K.
Wendel
 
U.
Janssen
 
B.
 
A patient with α-ketoadipic and α-aminoadipic aciduria
J. Inherit. Metab. Dis.
1984
, vol. 
7
 pg. 
61
 
Manders
 
A. J.
von Oostrom
 
C. G.
Trijbels
 
J. M.
Rutten
 
F. J.
Kleijer
 
W. J.
 
α-Aminoadipic aciduria and persistence of fetal haemoglobin in an oligophrenic child
Eur. J. Pediatr.
1981
, vol. 
136
 (pg. 
51
-
55
)
Rhee
 
S.
Silva
 
M. M.
Hyde
 
C. C.
Rogers
 
P. H.
Metzler
 
C. M.
Metzler
 
D. E.
Arnone
 
A.
 
Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
17293
-
17302
)
Miyahara
 
I.
Hirotsu
 
K.
Hayashi
 
H.
Kagamiyama
 
H.
 
X-ray crystallographic study of pyridoxamine 5′-phosphate-type aspartate aminotransferases from Escherichia coli in three forms
J. Biochem.
1994
, vol. 
116
 (pg. 
1001
-
1012
)
Malashkevich
 
V. N.
Strokopytov
 
B. V.
Borisov
 
V. V.
Dauter
 
Z.
Wilson
 
K. S.
Torchinsky
 
Y. M.
 
Crystal structure of the closed form of chicken cytosolic aspartate aminotransferase at 1.9 Â resolution
J. Mol. Biol.
1995
, vol. 
247
 (pg. 
111
-
124
)
McPhalen
 
C. A.
Vincent
 
M. G.
Jansonius
 
J. N.
 
X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase
J. Mol. Biol.
1992
, vol. 
225
 (pg. 
495
-
517
)
Okamoto
 
A.
Higuchi
 
T.
Hirotsu
 
K.
Kuramitsu
 
S.
Kagamiyama
 
H.
 
X-ray crystallographic study of pyridoxal 5′-phosphate-type aspartate aminotransferases from Escherichia coli in open and closed form
J. Biochem.
1994
, vol. 
116
 (pg. 
95
-
107
)
Jager
 
J.
Moser
 
M.
Sauder
 
U.
Jansonius
 
J. N.
 
Crystal structures of Escherichia coli aspartate aminotransferase in two conformations. Comparison of an unliganded open and two liganded closed forms
J. Mol. Biol.
1994
, vol. 
239
 (pg. 
285
-
305
)
Han
 
Q.
Gao
 
Y. G.
Robinson
 
H.
Li
 
J.
 
Structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase
Biochemistry
2008
, vol. 
47
 (pg. 
1622
-
1630
)

Author notes

The structural co-ordinates reported will appear in the Protein Data Bank under accession code 3DC1.