Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine...

Biosci. Rep. (2008) / 28 / 205–215 (Printed in Great Britain) / doi 10.1042/BSR20080085

Substrate specificity and structure of humanaminoadipate aminotransferase/kynurenineaminotransferase IIQian HAN*1, Tao CAI†, Danilo A. TAGLE‡, Howard ROBINSON§ and Jianyong LI*

*Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, U.S.A., †OIIB, NIDCR (National Institute of Dental and CraniofacialResearch), National Institutes of Health, Bethesda, MD 20892-4322, U.S.A., ‡Neuroscience Center, NINDS (National Institute ofNeurological Disorders and Stroke), National Institutes of Health, Bethesda, MD 20892-9525, U.S.A., and §Biology Department,Brookhaven National Laboratory, Upton, NY 11973, U.S.A.

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SynopsisKAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenineto 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 namedAADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergicneurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical andfunctional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressingits substrate profile and kinetic properties has not been performed. The present study examines the biochemicaland structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that theenzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 testedamino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KATII demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as toits preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed aconformational 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.

Key words: aminoadipic acid, crystal structure, kynurenic acid (KYNA), kynurenine, kynurenine aminotransferase(KAT), neurodegenerative disease

INTRODUCTION

Aminotransferase, capable of catalysing the transamination ofkynurenine to KYNA (kynurenic acid), has commonly beentermed KAT (kynurenine aminotransferase). KYNA is the onlyknown endogenous antagonist of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptors [1–4]. KYNA is alsothe antagonist of the α7-nicotinic acetylcholine receptor [5–8].The level of KYNA is altered in several neurodegenerative dis-eases, including Huntington’s disease [9,10], Alzheimer’s disease[11], schizophrenia [12–14] and acquired immunodeficiency syn-drome dementia [15]. Because KYNA must be produced byKAT-catalysed kynurenine transamination, aminotransferases,responsible for catalysing kynurenine to KYNA, have been con-

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Abbreviations used: AADAT, aminoadipate aminotransferase; KAT, kynurenine aminotransferase; hKAT II, human kynurenine aminotransferase II; KYNA, kynurenic acid; LLP, lysinepyridoxal-5′ -phosphate; NMDA, N-methyl-D-aspartate; OPT, O-phthaldialdehyde thiol; PLP, pyridoxal 5′ -phosphate.The structural co-ordinates reported will appear in the Protein Data Bank under accession code 3DC1.1To whom correspondence should be addressed (email [email protected]).

sidered to be targets for maintaining and regulating physiologicalconcentrations 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 centralnervous system [16–21]. Of these, KAT I and KAT II have beenextensively studied and the crystal structures of hKAT I (humankynurenine aminotransferase I) and hKAT II (human kynurenineaminotransferase II) are now available [16,17,19,22–25]. It hasbeen reported that KAT I has a broad substrate specificity anddisplays maximum activity at relatively basic conditions. Thisposes critical questions regarding the contribution of KAT I tobrain KYNA production [16,17]. On the other hand, KAT II isidentical to AADAT (aminoadipate aminotransferase) and cata-lyses the transamination of kynurenine and aminoadipate and islocalized in the soluble cytoplasm [16]. The enzyme has been

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Q. Han and others

cloned from both rats and humans and its presence in the brainhas been confirmed by Northern and Western blotting [26–29].Previous studies on KAT II/AADAT have been focused on itsactivity on aminoadipate in peripheral organs, especially in theliver and the kidney, and on its role in brain KYNA production[30–32].

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

MATERIALS AND METHODS

Expression, purification and activity assayof recombinant hKAT IIProtein expression and purificationRecombinant hKAT II coding sequence (GenBank® NucleotideSequence accession number NM_182662) was cloned into anImpactTM-CN plasmid (New England Biolabs) for expression ofa fusion protein containing a chitin-binding domain. hKAT II waspurified by affinity purification, DEAE–Sepharose, hydroxyapat-ite and gel-filtration chromatography. The purified recombinanthKAT II was concentrated to 10 mg · ml−1 of protein in 10 mMphosphate buffer (pH 7.4) using a Centricon YM-30 concentrator(Millipore) [24].

Activity assayThe KAT activity assay was based upon methods described pre-viously [33]. Briefly, a reaction mixture (50 μl final volume)containing 10 mM L-kynurenine, 2 mM α-oxoglutarate, 40 μMPLP (pyridoxal 5′-phosphate) and 2 μg of the protein sample wasprepared in 100 mM potassium phosphate buffer (pH 7.4). Themixture was incubated at 37◦C for 15 min, and the reaction wasstopped by the addition of an equal volume of 0.8 M formic acid.The supernatant of the reaction mixture, obtained by centrifu-gation at 15 000 g for 10 min at 4◦C, was analysed by HPLC,with UV detection performed at 330 nm for both kynurenine andKYNA.

Effect of pH and temperature on hKAT IITo determine the effect of the buffer pH on hKAT II activity,a buffer mixture consisting of 100 mM phosphate and 100 mMboric acid was prepared, and the pH of the buffer was adjusted topH 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0. The buffer mixture was se-lected to maintain a relatively consistent buffer composition andionic strength, but would have sufficient buffering capacity for arelatively broad range of pH values. A typical reaction mixturecontaining 10 mM kynurenine, 2 mM α-oxoglutarate and 2 μg ofhKAT II was prepared using the buffer mixture at different pHs.

The reaction mixture was incubated and analysed in the hKATII activity assay as described above. To determine the effect oftemperature on hKAT II-catalysed kynurenine transamination,the typical reaction mixture [final volume of 50 μl in 100 mMphosphate buffer (pH 7.4)] was incubated at temperatures ran-ging from 10–80◦C for 15 min and the product formed in themixture was analysed as described above for the hKAT II activityassay.

Substrate specificityTo test the transamination activity of other amino acids, an as-say mixture containing 15 mM amino acid, 20 mM glyoxylateor α-oxobutyrate (for glycine), 40 μM PLP, 100 mM potassiumphosphate buffer (pH 7.4) and 2 μg of enzyme was prepared in atotal volume of 50 μl. The mixture was incubated for 15 min at37◦C. The product was quantified based on the detection of theOPT (O-phthaldialdehyde thiol)–amino-acid product conjugateby HPLC, with electrochemical detection performed after theircorresponding reaction mixtures were derivatized using an OPTreagent [34]. In a kinetic study for amino-acid substrates, the typ-ical reaction mixture specified in the KAT activity assay [50 μlfinal volume prepared in 100 mM phosphate buffer (pH 7.4)]containing various concentrations (0.47–30 mM) of the aminoacids which showed detectable transaminase activity with hKATII and 30 mM glyoxylate was used. The mixture was incubatedfor 15 min at 37◦C. To determine the substrate specificity forα-oxo acids, 16 individual α-oxo acids were tested for their abil-ity to function as the amino-group acceptor for hKAT II. In theassays, the typical reaction mixture stated above also containeda different α-oxo acid at various concentrations (0.47–60 mM)and 15 mM kynurenine, and the rate of KYNA production wasdetermined by performing the KAT activity assay as describedabove. The kinetic parameters were calculated by fitting theMichaelis–Menten equation to the experimental data using theenzyme kinetics module in SigmaPlot 8.2 (SPSS).

The effect of other amino acids on KAT enzyme activityAnalysis of the substrate specificity revealed that a number ofamino acids could serve as the amino-group donor for hKAT II.To determine the effect of other amino acids on hKAT II-catalysedKYNA production, a different amino acid (final concentration of5 mM) was incorporated into the typical reaction mixture (totalvolume of 50 μl) which contained 5 mM kynurenine, 20 mMglyoxylate and 2 μg of hKAT II (pH 7.4) and the enzyme activitywas assayed in the same manner as described above for the KATactivity assay.

Crystal structure of the hKAT II complexwith α-oxoglutaric acidThe crystals were grown using the hanging-drop vapour diffusionmethods described previously [24]. The crystals of the enzyme–α-oxoglutaric acid complex were obtained by soaking the crys-tals in 2.5 mM α-oxoglutaric acid in the crystallization bufferfor 3 days. Individual hKAT II complex crystals were cryogen-ized using 25 % (v/v) glycerol in the crystallization buffer as a

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206 C©The Authors Journal compilation C©2008 Biochemical Society

Human aminoadipate aminotransferase/kynurenine aminotransferase II

Table 1 Data collection and refinement statistics of the hKAT IIcomplex with α-oxoglutaric acidGOL, glycerol; 2OG, α-oxooglutaric acid; RMS, root mean square.

Crystal data

Space group P1

Unit cell

a (A◦) 60.9

b (A◦) 72.1

c (A◦) 109.1

α (◦) 90.0

β (◦) 100.7

γ (◦) 93.9

Data collection

X-ray source Brookhaven National

Laboratory-X29

Wavelength (A◦) 1.0809

Resolution (A◦)* 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 (A◦) 0.020

RMS bond angles (◦) 1.857

Number of ligand or 4 LLP

cofactor molecules

1 2OG

7 GOL

Number of water molecules 828

Average B overall (A◦2) 33.9

* The values in parentheses are for the highest resolution shell.

cryoprotectant solution. Diffraction data for the crystal were col-lected at the Brookhaven National Synchrotron Light Sourcebeam line X29A [λ = 1.0908 A (1 A = 0.1 nm)]. All data were in-dexed and integrated using HKL-2000 software (HKL Research),and scaling and merging of the diffraction data were performedusing the program SCALEPACK [35]. The parameters of thecrystals and data collection are listed in Table 1. The structureof hKAT II was determined using the molecular-replacementmethod using the hKAT II structure (Protein Data Bank code2QLR). The program Molrep [36] was employed to calculateboth cross-rotation and translation functions of the model. Theinitial model was subjected to iterative cycles of crystallographicrefinement with the Refmac 5.2 [37] and graphic sessions formodel building using the program O [38]. Solvent molecules wereautomatically added and refined with ARP/wARP [39] togetherwith Refmac 5.2. Superposition of the structures was done usingLsqkab [40] in the CCP4 suite, and Figures were generatedusing PyMOL [41].

Figure 1 Effect of pH and temperature on enzyme activityThe activities of recombinant hKAT II at different temperatures (A) andpH values (B). T, temperature. Results are means +− S.D. (n = 2).

RESULTS

Biophysical properties of hKAT IIOn the basis of SDS/PAGE analysis, very high purity of the isol-ated hKAT II was achieved. The isolated hKAT II was stableunder freezing conditions (results not shown). No noticeable de-crease in specific activity was observed when aliquots of hKATII were stored at −80◦C during a period of 6 months, which al-lowed us to analyse critically the substrate specificity and kineticproperties of hKAT II using aliquots of the enzyme from the samebatch of highly purified hKAT II. The enzyme showed maximumactivity at approx. 50◦C and had a broad optimum pH range frompH 7–9 (Figure 1), which is similar to those reported previously[16,17].

Substrate study of hKAT IIhKAT II was tested for activity towards 24 amino acids and 16oxo acids. Of the 24 amino acids tested, hKAT II was able tocatalyse transamination of 20 of the amino acids. The specificactivity of hKAT II towards individual amino acids under theconditions applied is shown in Figure 2. This substrate screening

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Q. Han and others

Figure 2 Effect of other amino acids on hKAT II/KAT activityThe activities were quantified by measuing the amount of KYNA produced in the reaction mixtures. The rate of KYNA pro-duction 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.

assay determined that hKAT II has a very broad substratespecificity. Kinetic analysis of hKAT II resulted in the determina-tion of the catalytic efficiency of the enzyme towards individualamino-group donors and acceptors (Table 2), which provideda basis to determine its primary amino-group donors and ac-ceptors and for speculation regarding its primary physiologicalfunctions. On the basis of the kinetic data, hKAT II should beequally efficient in catalysing the transamination of aminoad-ipate, kynurenine, methionine and glutamate (Table 2). All 16α-oxo acids tested were capable of serving as amino-group ac-ceptors. Of these, α-oxoglutarate, α-oxocaproic acid, phenylpyr-uvate and α-KMB (α-oxo-γ-methiol-butyric acid) were highlyefficient as amino-group acceptors for hKAT II (Table 2). Al-though glyoxylate showed a relatively low affinity for hKAT II,it did not inhibit hKAT activity at high concentrations and itsglycine–OPT complex was easily resolved from all other aminoacid–OPT complexes during HPLC analysis with electrochem-ical detection. Therefore glyoxylate has been used as the primaryamino-group acceptor for screening of the amino-group donorsof hKAT II and kinetic analysis of the enzyme in the presence ofdifferent amino-group donors. KYNA displayed an absorbancepeak with a λmax of approx. 330 nm and was easily resolved fromkynurenine during reverse-phase separation; therefore kynuren-ine was used as the primary amino-group donor during screeningof amino-group acceptors and their kinetic analysis.

Effects of other amino acids on hKAT II-catalysedkynurenine transaminationOn 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 the23 amino acids (at 5 mM concentration) was incorporated with5 mM kynurenine into the kynurenine/hKAT II/α-oxo acid re-action mixture, aminoadipate, asparagine, glutamate, histidine,cysteine, lysine, 3-hydroxy-kynurenine and phenylalanine wereall shown to decrease the rate of KYNA formation, but none ofthem inhibited more than 35 % of KAT activity (Figure 2).

Overall structureThe crystal structure of the hKAT II–α-oxoglutaric acid complexwas determined by molecular replacement and refined to a resolu-tion of 2.48 A. The final model yields a crystallographic R value of21.5 % and an Rfree value of 24.4 % with ideal geometry (Table 1).There are four protein molecules that form two biological ho-modimers in an asymmetric unit and the protein residues arenumbered 1(A)–425(A) for molecule A, 1(B)–425(B) for mol-ecule B, 1(C)–425(C) for molecule C, and 1(D)–425(D) formolecule D. Pro18 (A) to Gly29 (A), Arg20 (B) to Gly29 (B), andArg20 (D) to Arg28 (D) have not been included in the final hKATII–α-oxoglutaric acid complex model because they are highlydisordered. To distinguish the residues from the two subunits ofa biological dimer in the description of the substrate interaction,residues from the opposite subunit have been labelled with an as-terisk (*). The results of the refinement are summarized in Table 1.

The structural basis of the broad substratespecificity of hKAT IIWe were able to model α-oxoglutaric acid in one of the four sub-units. In the other three subunits, the equivalent position is occu-pied by glycerol. Inspection of the active centre of the hKAT II

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Table 2 Kinetic parameters of hKAT II towards amino acids and α-oxo acidsThe Km and kcat for oxo acids were derived by using various concentrations of individual oxo acids in thepresence of 10 mM kynurenine; for amino acids, the results were derived by using various concentrationsof 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

crystal structure revealed that the substrate lies near the PLPmolecule that forms LLP (lysine pyridoxal-5′-phosphate) withLys263. Several residues, including Tyr142 and Asn202 from onesubunit, and Gly39* and Tyr74* from the opposite subunit, definethe substrate-binding site and contact the α-oxoglutaric acid mo-lecule. The ring of Tyr74* has a weak hydrophobic interactionwith α-oxoglutaric acid, and its hydroxy group forms a weakhydrogen bond with the O2 atom of α-oxoglutaric acid (Fig-ure 3). The interactions of glycerol and the hKAT II active-centreresidues include the formation of hydrogen bonds between gly-cerol and residues Tyr142, Gln118 and Tyr233 and hydrophobic in-teractions between glycerol and residues Tyr74* and Leu19* fromthe opposite subunit (Figure 4). Upon superposing of the hKAT IIsubunit B (with α-oxoglutaric acid) and hKAT II subunit D (withglycerol) on to the hKAT II–kynurenine structure (Figure 5), weidentified the residues within 5 A of the ligands. α-Oxoglutaricacid, glycerol and kynurenine basically occupy similar positions,

but α-oxoglutaric acid has fewer interactions with the enzymethan kynurenine. The carboxylic group of the α-oxoglutaric acidsubstrate does not form salt bridges with the guanidinium groupof Arg399. PLP forms LLP with Lys263 when the subunit bindsglycerol or α-oxoglutaric acid, and forms PMP (pyridoximine5′-phosphate) when the protein binds kynurenine. Most residueswithin 5 A have the same conformation, except for a few residuesin the N-terminal fragment (residues 15–33) (Figure 5). When theprotein molecule binds glycerol, this N-terminal fragment movestowards the centre to interact with glycerol. However, when itbinds kynurenine, the N-terminal fragment moves away from thecentre and leaves room for kynurenine. Also, when it binds α-oxoglutaric acid, the N-terminal fragment seems to move furtheraway to leave more space for the substrate. In summary, the find-ing that the N-terminal fragment is disordered simply indicatesthat the N-terminal fragment is highly flexible and is moved uponbinding of the substrate.

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Q. Han and others

Figure 3 α-Oxoglutaric acid binding siteα-Oxoglutaric acid (2OG) and the protein residues within a distance of 4 A

◦of 2OG are shown (distances are stated in A

◦).

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 4 Glycerol-binding site in the active centreGlycerol (GOL) and the protein residues within a 4 A

◦distance of GOL are shown (distances are stated in A

◦). The 2Fo-Fc

electron-density map covering GOL is contoured at the 1.1σ level. The Figure was generated by PyMOL. * indicates residueson the opposite subunit.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



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 areshown in ribbon form (residues 15–33). GOL, glycerol; Kyn, kynurenine;LLP263, PLP formed with Lys263; 2OG, α-oxoglutaric acid, PMP, pyridox-imine 5′ -phosphate.

DISCUSSION

AADAT was first detected in rat liver [42] and its potential func-tion in liver lysine degradation has been well described [43]. Itwas reported that the same aminotransferase from liver and kid-ney had both KAT activity and AADAT activity [44–46] and thetrue identity of rat KAT II and AADAT was confirmed by Tobesand Mason [45].

KAT II has been considered to be the principal enzyme re-sponsible for the synthesis of KYNA in the rodent and humanbrain [17,18]. Mice in which KAT II was knocked out by gen-omic manipulation showed a substantial decrease in brain KYNAlevels and displayed phenotypic changes that were in line withthis chemical deficit [7,47,48]. In the present study, kinetic ana-lysis shows a better overall catalytic efficiency of hKAT II towardskynurenine than towards other amino-group donors, except foraminoadipate. Although hKAT II has a relatively high Km forkynurenine, its catalytic efficiency in the kynurenine to KYNApathway is enhanced by its relatively high catalytic-centre activ-ity towards kynurenine and also by the irreversible nature ofthe kynurenine to KYNA pathway. The enzymatic productof kynurenine transamination is an α-oxo acid intermediate thatis unstable and undergoes rapid intramolecular cyclization toKYNA. Consequently, no direct enzymatic product accumulatesduring KAT-catalysed kynurenine transamination and the equi-librium always proceeds towards KYNA production. Thereforethe transamination of kynurenine to KYNA is one of the majorfunctions of hKAT II.

Although most of the previous studies on KAT II have emphas-ized its function in KYNA biosynthesis because of the important

Figure 6 Lysine catabolism pathway in mammals

role of KYNA in the central nervous system, the enzyme hasa higher catalytic efficiency for aminoadipate than for kynuren-ine (Table 2). Human liver has been shown to have AADATactivity [49]. In rats, AADAT activity has been found in liverand kidney tissues [42,44–46,50–52]. In humans, lysine is themain source of aminoadipate. Lysine, an essential amino acidin humans, is degraded when there is an excess present beyondthat required for protein synthesis. The aminoadipate pathwayis the major route for the catabolism of lysine and occurs inthe liver [27,53,54]. From the enzyme kinetic parameters in thispaper, we showed that aminoadipate was the best amino-acidsubstrate for hKAT II. The fact that α-oxoadipate is not a goodamino-group acceptor for the enzyme suggests that the AADATenzyme reaction does not tend to be reversible, and favours thedirection from aminoadipate to α-oxoadipate, one of the reac-tions involved in lysine degradation (Figure 6). An additionalargument for a primary role of hKAT II in the aminoadipate toα-oxoadipate pathway comes from the comparison of hKAT IIwith its homologues from some micro-organisms. The KAT II orAADAT present in lower eukaryotes and certain bacteria is in-volved primarily in lysine biosynthesis (the reversal of lysinecatabolism) because it catalyses the transamination reaction us-ing α-oxoadipate as an amino acceptor to yield aminoadipate[55–58]. The kinetic constant (Km) of a yeast KAT II homologueis 14 mM for α-oxoadipate and 20 mM for aminoadipate [59].The homologue enzyme from Thermus thermophilus catalyses

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Q. Han and others

efficiently the transamination from α-oxoadipate to aminoadip-ate with negligible activity in the reverse reaction [55]. Theseresults show that the KAT II homologues in yeast and T. ther-mophilus favour the transamination direction from α-oxoadipateto aminoadipate, the reaction involved in lysine biosynthesis. Incontrast, 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.

Aminoadipate occurs naturally in the brain [53,60]. The com-pound is known to be a selective toxin for astrocytes and relatedglial cells because injection of a racemic mixture of aminoadip-ate induced transient degenerative changes in non-neuronal cells[61]. Its toxicity to glial cells has been further demonstrated bya number of subsequent in vivo or in vitro studies [62–69]. SinceKAT II is present in the astrocyte-like cells of mammalian brains[28,70,71], it raises a reasonable argument for the role of KAT IIin the neutralization of aminoadipate in the mammalian brain. Onthe other hand, many previous studies have shown that a high con-centration of aminoadipate inhibits glutamate transport [72,73],blocks glutamine synthetase [74], prevents the uptake of glutam-ate into synaptic vesicles [75] and functions as an NMDA receptoragonist [76]. These effects can contribute to an increased excitat-ory tone because synaptic glutamate concentrations are elevated.Therefore the hyperactive behaviour observed in katII−/− mice[47] raised an interesting question as to whether the aminoadipatelevel is increased in the knockout mouse brain and consequentlycontributes to the pathological mechanisms of the abnormal be-haviour. High levels of aminoadipate in serum and/or urine havealso been observed in many cases with neurological and otherdisorders [77–87].

Biochemical analysis indicates that hKAT II is a multifunc-tional aminotransferase. Kinetic analysis of the enzyme towardsdifferent amino acids and oxo acids showed that the enzymewas efficient in catalysing the transamination of a number ofother amino acids, as well as kynurenine and α-aminoadipate,and could use many α-oxo acids as amino-group acceptors. Thecharacterization of multiple substrates for hKAT II suggests thatthe enzyme might play a role in the metabolism of other oxo acidsand amino acids, which requires further study. It also raises a fun-damental question regarding the structural basis of its broad sub-strate specificity. A previous study [24] showed that a major con-formational change of residues 16–31 in hKAT II is involved insubstrate binding and for shielding the substrate-binding pocketfrom bulk solvents. The hKAT II–α-oxoglutaric acid structureshows that this N-terminal fraction is very flexible and it hasdifferent conformations when the protein subunit binds to differ-ent ligands. This N-terminal fraction moves close to the activecentre when the protein subunit binds a small ligand, and movesaway from the centre when the subunit binds a large ligand. Inthe hKAT II–α-oxoaglutarate structure we did not find any otherlarge-scale conformational changes, such as domain–domain rot-ation, which is involved in binding substrates in other amino-transferases [88–94]. So the conformational change of part ofthe N-terminal residues is the main mechanism which adapts toaccommodate various substrates of different sizes in the activecentre.

In summary, there have been a number of previous studies[16,17,44–46] dealing with the biochemical characterization ofKAT II/AADAT, which emphasize either the KAT activity of theenzyme or its AADAT function. The present study, however, pro-duces an overall picture regarding the substrate profiles of hKATII, its catalytic efficiency for different amino-group donors andacceptors and its structural basis of substrate binding and cata-lysis. Our results suggest that hKAT II is likely to play primaryroles in both the kynurenine to KYNA and aminoadipate to α-oxoadipate pathways. In addition, the enzyme may be involvedin the regulation of other amino acids and α-oxo acids. Thereforeit will be interesting to study the overall functions of hKAT II invivo in the future, particularly in order to use the available Kat IIgene-deficient mice to test any possible hypotheses.

ACKNOWLEDGEMENTS

This work was carried out in part at the National SynchrotronLight Source, Brookhaven National Laboratory (Upton, NY 11973,U.S.A.). This research was supported in part by the Intramural Re-search Program of the institutes of the NIDCR and NINDS at theNIH (National Institutes of Health).

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Received 3 July 2008; accepted 14 July 2008Published as Immediate Publication 14 July 2008, doi 10.1042/BSR20080085

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Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine...

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