Biosci. Rep. (2011) / 31 / 323–332 (Printed in Great Britain) / doi 10.1042/BSR20100117

Biochemical and structural characterization ofmouse mitochondrial aspartateaminotransferase, a newly identified kynurenineaminotransferase-IVQian HAN*, Howard ROBINSON†, Tao CAI‡, Danilo A. TAGLE§ and Jianyong LI*1

*Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, U.S.A., †Biology Department, Brookhaven National Laboratory, Upton,NY 11973, U.S.A., ‡Oral Infection and Immunity Branch (OIIB), National Institute of Dental and Craniofacial Research (NIDCR), NationalInstitutes of Health, Bethesda, MD 20892-4322, U.S.A., and §Neuroscience Center, National Institute of Neurological Disorders and Stroke(NINDS), National Institutes of Health, Bethesda, MD 2089-29525, U.S.A.

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SynopsisMammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine amino-transferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains.This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspATcDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichiacoli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT bymacromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of otherKATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide abiochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insightinto controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

Key words: aspartate aminotransferase, crystal structure, oxo acid, kynurenic acid, kynurenine, kynurenineaminotransferase

INTRODUCTION

KYNA (kynurenic acid) is the only known endogenous antagon-ist of the NMDA (N-methyl-D-aspartate) subtype of glutamatereceptors [1–4]. KYNA is also the antagonist of the α7-nicotinicacetylcholine receptor [5–8]. In mammalian brains, glutam-ate is the major excitatory neurotransmitter and acts throughboth ligand-gated ion channels and G-protein-coupled receptors,which are collectively called glutamate receptors [9]. Activationof these receptors is responsible for basal excitatory synaptictransmission and many forms of synaptic plasticity such as long-term potentiation and long-term depression, which are thoughtto underlie learning and memory [9,10]. However, any event orprocess leading to a sudden or chronic increase in the activity ofglutamate receptors often induces the death of neurons [11]. Con-

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Abbreviations used: CCBL, cysteine conjugate β -lyase; FABP, fatty acid binding protein; GPR35, G-protein-coupled receptor 35; KAT, kynurenine aminotransferase; αKMB,α-oxo-γ -methiobutyric acid; KYNA, kynurenic acid; mAspAT, mitochondrial aspartate aminotransferase; PLP, pyridoxal-5′ -phosphate; PMP, pyridoximine 5′ -phosphate.1To whom correspondence should be addressed (email: lij@vt.edu).The structural co-ordinates reported will appear in the PDB under accession codes 3PD6 and 3PDB.

sequently, a mechanism capable of preventing glutamate recept-ors from being overly stimulated seems essential for maintainingthe normal physiological condition of the brain [12,13]. BrainKYNA levels are abnormal in the progression of some neur-ological and psychiatric disorders (see review in [14]), whichsuggests that variations in brain KYNA, acting as an endogenousmodulator of glutamatergic and cholinergic neurotransmission,may be functionally significant.

In addition to the roles KYNA plays in the central nervoussystem, KYNA has been identified as an agonist for the previ-ously ‘orphaned’ receptor GPR35 (G-protein-coupled receptor35) [15]. More recently, a study has demonstrated that KYNA isimplicated in the regulation of leukocyte binding on the endothe-lium due to the activation of GPR35 by KYNA [16].

KYNA is produced enzymatically by the irreversible trans-amination of kynurenine, the key intermediate in the tryptophan

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catabolic pathway. In humans, rats and mice, four proteins arbit-rarily named KAT (kynurenine aminotransferase) I, II, III and IVhave been considered to be involved in KYNA biosynthesis in thecentral nervous system [17–23]. KAT I is identical with glutam-ine transaminase K and CCBL (cysteine conjugate β-lyase) 1;KAT II is identical with aminoadipate aminotransferase; KATIII is identical with CCBL 2; and KAT IV is identical withglutamic-oxaloacetic transaminase 2 or mAspAT (mitochondrialaspartate aminotransferase). These proteins are all PLP (pyri-doxal 5′-phosphate)-dependent enzymes. Among the individualmammalian KATs, KAT I [20,24–26] and KAT III [23] sharesimilar genomic structure and high-sequence identity [21] andhave been assigned to subgroup Iγ in fold type I aminotrans-ferases [14,27]. KAT II is unique in swapping the catalyticallyessential N-terminal region [28–30]. A further sequence phylo-genetic analysis revealed that KAT II and KAT II homologuesform a separate lineage [31]. These indicate that KAT II and itshomologues actually form a new subgroup in fold type I amino-transferases, designated as subgroup Iε, the eighth subgroup inthe fold type [14]. Based on the sequence information, KATIV/mAspAT has been assigned to subgroup Iα [27]. AspATsfrom different sources have been used as a model for studyingthe mechanism of catalysis for aminotransferases and hundredsof AspAT structures are available in the PDB. Among these,the only mitochondrial AspAT structures available are from thechicken [32]; there are none from mammals or humans. It hasbeen reported that mAspAT/KAT IV plays a major role in KYNAsynthesis in mouse brain [22]. A recent biochemical test supportsthis assertion [33], indicating that mAspAT/KAT IV could be aprimary target for brain KYNA regulation. In the present study,we report the crystal structure of mouse mAspAT in its PLP formas well as its structures in complex with substrate kynurenine andco-substrate oxaloacetate. We also provide a kinetic characteriz-ation of mAspAT with 12 co-substrates. Determination of thethree-dimensional structure of mouse mAspAT may contributeto the rational design of selective inhibitors that are of intensemedical interest with respect to a number of human pathologicalconditions in which the brain KYNA level is abnormal.

EXPERIMENTAL

Expression and purification of recombinant mAspATmAspAT coding sequence (GenBank® accession no.NM_010325) was amplified from a mouse brain cDNApool and cloned into an ImpactTM-CN plasmid (New EnglandBiolabs) for expression of a fusion protein containing achitin-binding domain. Transformed Escherichia coli cellswere cultured and harvested as the start materials for affinitypurification. Further purification of the recombinant mAspATwas achieved by ion exchange (Q-Sepharose) and gel filtrationchromatographies. The purified recombinant mAspAT was con-centrated to 10 mg of protein ml− 1 in 10 mM phosphate buffer(pH 7.5) using a Centricon YM-30 concentrator (Millipore) [33].

KAT activity assayKAT activity assay was based on previously described meth-ods [20,23,29]. Briefly, a reaction mixture of 100 μl, containing5 mM L-kynurenine, 2 mM α-oxoglutarate, 40 μM PLP and 5 μgof recombinant protein, was prepared using 100 mM potassiumphosphate buffer (pH 7.5). This reaction mixture is identifiedhereafter as the typical reaction mixture. The mixture was incub-ated for 15 min at 38◦C and the reaction stopped by adding anequal volume of 0.8 M formic acid. The supernatant of the reac-tion mixture, obtained by centrifugation at 15 000 g for 10 minat room temperature, was analysed for the product, KYNA, byHPLC with UV detection at 330 nm.

Co-substrate specificityTo determine the substrate specificity for α-oxo acids, 16 α-oxo acids were individually tested for their ability to func-tion as an amino group acceptor for mouse mAspAT. Eachof the 16 α-oxo acids was assayed at 2 mM in the presence of5 mM kynurenine in the 100 μl typical reaction mixture andthe rate of KYNA production was determined as describedin the KAT activity assay. The kinetic study for α-oxo acidsubstrates of mouse mAspAT is based on a previously usedmethod [23].

Mouse mAspAT crystallizationThe crystals were grown by the hanging-drop vapour diffusionmethod with the volume of reservoir solution at 500 μl and thedrop volume at 2 μl, containing 1 μl of protein sample and 1 μlof reservoir solution. The optimized crystallization buffer con-sisted of 20 % poly(ethylene glycol) 4000, 100 mM ammoniumsulfate and 6 % glycerol. mAspAT–kynureine complex crystalswere prepared by co-crystallizing the enzyme in the presence of2 mM kynurenine, and mAspAT–oxaloacetate complex crystalsin the presence of 2 mM oxaloacetate (previously neutralized byNaOH).

Data collection and processingIndividual mouse mAspAT crystals were cryogenized using 20 %glycerol in the crystallization buffer as a cryo-protectant solution.Diffraction data of mouse mAspAT crystal were collected at theBrookhaven National Synchrotron Light Source beam line X29A(λ = 1.0908 A). Data were collected using an ADSC Q315 CCDdetector. All data were indexed and integrated using HKL-2000software; scaling and merging of diffraction data were performedusing the program SCALEPACK [34]. The parameters of thecrystals and data collection are listed in Table 1.

Structure determinationThe structure of mouse mAspAT was determined by the molecu-lar replacement method using the published chicken mAspAT(PDB accession number 7AAT) [32]. The program Molrep [35]was employed to calculate both cross-rotation and translation

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Table 1 Data collection and refinement statisticsGOL, glycerol; KYN, kynurenine; BME, 2-mercaptoethanol; BNL, Brookhaven National Laboratory; PMP, pyridoximine5′ -phosphate.

Parameter mAspAT mAspAT–oxaloacetate mAspAT–KYN

Crystal data

Space group P21212

Unit cell

a (A) 76.8 77.9 282.4

b (A) 87.4 87.4 78.1

c (A) 284.3 282.4 87.5

α = β = γ (◦) 90.0 90.0 90

Data collection

X-ray source BNL-X29

Wavelength (A) 1.0809

Resolution (A)* 2.50 (2.59–2.50) 2.40 (2.49–2.40) 2.40 (2.49–2.40)

Total number of reflections 751, 633 730, 307 585, 440

Number of unique reflections 67, 188 76, 929 76, 708

R-merge* 0.12 (0.42) 0.12 (0.32) 0.11 (0.34)

Redundancy* 12.8 (3.8) 10.3 (3.7) 8.1 (2.3)

Completeness (%)* 92.4 (54.9) 92.3 (55.3) 93.9 (60.4)

Refinement statistics

R-work (%) 18.1 17.7 17.8

R-free (%) 23.4 19.5 18.9

RMS bond lengths (A) 0.026 0.016 0.019

RMS bond angles (◦) 1.982 1.597 1.725

Number of ligand or cofactor molecules 4 LLP 2 LLP 2 LLP

6 GOL 5 GOL 13 GOL

1 oxaloacetate

7 BME 2 PMP

2 PMP 1 KYN

Number of water molecules 689 721 831

Average B overall (A2) 39.1 28.3 29.1

Statistics on the Ramachandran plot (%)

Most favoured regions 89.1 91.6 91.4

Additional allowed regions 10.4 8.1 8.3

Generously allowed regions 0.2 0.0 0.1

Disallowed regions 0.3 0.3 0.3

∗The values in parentheses are for the highest-resolution shell.

functions of the model. The initial model was subjected to it-erative cycles of crystallographic refinement with the Refmac5.2 [36] and graphic sessions for model building using theprogram Coot [37]. Solvent molecules were automatically ad-ded and refined with ARP/wARP [38] together with Refmac5.2.

Analysis of biochemical data and crystal structureThe kinetic parameters of the recombinant enzyme towards dif-ferent α-oxo acids were calculated by fitting the Michaelis–Menten equation to the experimental data using the EnzymeKinetics Module for SigmaPlot (SPSS Science). Superpositionof structures was done using LSQKAB [39] in the CCP4 suite.

Figures were generated using PyMOL [40]. Protein and substrateinteraction were also analysed using PyMOL [40].

RESULTS

Co-substrate specificity of mouse mAspATMouse mAspAT was tested for KAT activity towards sixteen α-oxo acids using 5 mM kynurenine as the amino group donor.Fourteen of them had detectable activity (Figure 1). Table 2 illus-trates the enzyme kinetic parameters towards each α-oxo acid,including Km and kcat/Km. On the basis of kinetic analysis, mouse

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Table 2 Kinetic parameters of mouse mAspAT towards α-keto acidsThe activities were measured as described in the Experimental section. The Km and kcat for α-keto acids were derived by usingvarious concentrations (0.2–50 mM) of individual α-keto acids in the presence of 15 mM of kynurenine. The parameters werecalculated by fitting the Michaelis–Menten equation to the experimental data using the Enzyme Kinetics Module of SigmaPlot.Results shown are the means +− S.E.M.

Keto acid Km (mM) kcat (min− 1) kcat/Km (min− 1 · mM− 1)

Phenylpyruvate 0.7 +− 0.4 37.7 +− 6.9 57.8

Oxaloacetate 0.9 +− 0.4 19.2 +− 2.6 21.1

Hydroxyphenylpyruvate 1.6 +− 0.7 24.7 +− 5.5 15.7

α-Ketoglutarate 2.4 +− 0.6 32 +− 2.7 13.4

Mercaptopyruvate 3.2 +− 0.7 26.1 +− 1.6 8.1

Indo-3-pyruvate 3.6 +− 0.3 29.1 +− 1.1 8

αKMB 5.7 +− 0.5 24.8 +− 0.7 4.3

Glyoxylate 4.2 +− 0.8 11.4 +− 1.1 2.7

Pyruvate 8.3 +− 1.0 22.1 +− 0.9 2.7

α-Ketocaproic acid 10.4 +− 1.4 24.6 +− 1.2 2.4

α-Ketobutyrate 42.2 +− 14.2 41.3 +− 7.9 1

α-Ketovalerate 10.9 +− 1.5 7.5 +− 0.4 0.7

Figure 1 Transamination activity of mouse mAspAT towardsdifferent α-oxo acidsPurified recombinant mouse mAspAT was incubated with each of 16α-oxo acids at 2 mM in the presence of 5 mM kynurenine in a typical re-action mixture (100 mM phosphate, pH 7.5). The activity was quantifiedby the amount of KYNA produced in the reaction mixture.

mAspAT amino group acceptors with Km values less than 4 mMinclude phenylpyruvate, oxaloacetate, hydroxyphenylpyruvate,α-oxoglutarate, mercaptopyruvate and indo-3-pyruvate.

Overall structureThe structures of mouse mAspAT were determined by molecularreplacement and refined to 2.50 A resolution for the mAspAT PLPform, 2.40 A resolution for the mAspAT–kynurenine complexand 2.40 A resolution for the mAspAT–oxaloacetate complex.Final models contain 4×401 amino acid residues each and yield acrystallographic R value of 18.1 % and an Rfree value of 23.4 % for

its PLP form, 17.7 % and 19.5 % for the mAspAT–oxaloacetatecomplex, and 17.8 % and 18.9 % for the mAspAT–kynureninecomplex (Table 1). There are four protein molecules in an asym-metric unit, which form two biological homodimers. The residuesof the four subunits in mAspAT structures are numbered 30 (A)–430 (A) for chain A, 30 (B)–430 (B) for chain B, 30 (C)–430(C) for chain C and 30 (D)–430 (D) for chain D. The resultsof the refinement are summarized in Table 1. The statistics onRamachandran plot as defined with PROCHECK [41] is alsoshown in Table 1. An overview of the monomer structure modelis shown in Figure 2(A). The structure has an N-terminal arm(residues 30–42), large (residues 76–348) and small (residues43–75, 349–430) domains. The residue Asp243 interacts with thepyridine nitrogen of the cofactor, whose structural and functionalconservation in fold-type I of the PLP-dependent enzymes, indic-ates its importance for catalysis. Comparison analysis suggeststhat mAspATs share similar cofactor binding sites with the otherthree KATs.

Active site of mouse mAspATResidual electron density clearly revealed the presence of co-valently bound PLP in the cleft situated at the interface of thesubunits in the biological dimer of the PLP form of the structure.The C4A atom of PLP is covalently attached to the NZ atomof Lys279 through the formation of an internal Schiff base, andthe internal aldimine gives rise to residue LLP279, representedas sticks in Figure 3. The PLP pyridine ring is stacked betweenresidues Ala245 and Trp162 by hydrophobic interactions. The sidechains of Tyr246 and Asp243 are hydrogen bonded to O3 and N1of the pyridoxal respectively. The phosphate moiety of PLP isinteracting with Thr135, S133, Ser276, Arg287 and Tyr96 from theother subunit.

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Mouse mitochondrial aspartate aminotransferase/kynurenine aminotransferase-IV

Figure 2 Overall structure(A) Cartoon representation of the structure of mAspAT monomer. N-terminal arm (pink), small domain (blue) and largedomain (green) are shown in different colours. (B) On superposing all the subunits (11 monomers except for chain A inthe PLP form) in the three structures onto chain A in the structure of mAspAT PLP form, all the monomers are shownin ribbon. The fragment with significant conformation changes is indicated by an arrow.

Figure 3 mAspAT active siteThe PLP cofactor and the amino acid residues within a 4 A distance ofPLP are shown. The residues are coloured in teal (subunit A) and green(subunit B). Hydrogen bond lengths are labelled with the unit of A.

Substrate recognition and catalysisInspection of the crystal structure of the mAspAT–kynureninecomplex revealed that the substrate lies near the N1 atom ofpyridoximine 5′-phosphate, but kynurenine and the cofactor donot form an external aldimine. Several residues, including Ile44,Thr135, Trp162, Asn215, Arg287 and Arg407 from one subunit, andTyr96, Arg313, Ser317 and Asn318 from the other subunit, define thesubstrate-binding site and contact the kynurenine molecule. Thecarboxylic group of the kynurenine substrate forms a salt bridgewith the guanidinium group of Arg407. The salt bridge is fixed byhydrogen-bonding interactions with the side chain of Asn215 and

Gly65 at both sides of the salt bridge. The ring of Tyr96 (B) has aweak hydrophobic interaction with the phenyl ring of kynureninein this complex structure (Figure 4A).

mAspAT binds oxaloacetate in a similar manner in the crys-tal structure of the mAspAT–oxaloacetate complex as it bindskynurenine in the structure of mAspAT–kynurenine complexdescribed above. As the oxaloacetate molecule is smaller thankynurenine, there are fewer protein residues that interact with ox-aloacetate. The Thr135 and Arg287 residues from one subunit, andAsn318 from another subunit, all of which interact with kynuren-ine, are more than 4 A distance away from oxaloacetate andconsequently are not in close contact with it (Figure 4B).

Conformational changeOn superposing all the subunits (11 monomers except for chain Ain the PLP form) in three structures onto chain A in the structureof mAspAT PLP form, we identified that the large domains werewell superposed onto one another and the small domains were not,which suggests that conformational changes occur in the smalldomain (Figure 2A). The conformational changes of thesmall domain cause Ile44 and Arg407 to move and interact withthe substrate. This small domain conformational change not onlyfacilitates substrate binding but is also effective for shielding thesubstrate-binding pocket from bulk solvents. Carefully checkingthe conformation of substrate-binding residues, we determinedthat there were no significant side chain conformational changesof the substrate binding residues except for a slight change inresidue Arg313. It has been reported that by binding substrates,other AspATs and other aminotransferases change their conform-ations from an open to a closed form [14,23,32,42–47], whichinvolves a large-scale conformational change (domain–domainrotation). It seems that mouse mAspAT uses the same mechan-ism in substrate binding and catalysis.

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Figure 4 Substrate-binding site(A) Stereo view of the kynurenine-binding site in the mAspAT–kynurenine complex structure. The kynurenine (KYN) and theprotein residues within 4 A distance of the kynurenine are shown. The 2Fo − Fc electron density map covering thekynurenine is shown contoured at the 0.7 Sigma level. (B) Stereo view of the oxaloacetate (OAA) binding site inthe mAspAT–oxaloacetate complex structure. The oxaloacetate substrate (OAA) and the protein residues within 4 Adistance of the oxaloacetate substrate are shown. The 2Fo − Fc electron density map covering the oxaloacetate is showncontoured at the 0.9 sigma level.

DISCUSSION

Mouse mAspAT is a major aminotransferase involved in KYNAproduction in the brain; therefore, it can be considered as a po-tential regulatory target for maintaining physiological concentra-tions of brain KYNA. We report herein the co-substrate specificityand crystal structures of mouse mAspAT and its complexes withkynurenine and oxaloacetate. The enzyme can use a number ofα-oxo acids as co-substrates for KAT activity. The structuresof mAspAT PLP form and its complexes with kynurenine andoxaloacetate provide an important molecular basis for a com-prehensive understanding of the substrate binding and enzyme

catalysis in mAspAT, making it possible to work with structureand ligand-based design of the inhibitors of this enzyme.

There are four KATs in human and rodent brains [14]. Theidentification of a number of residues that are crucial for ligandbinding in the four KAT enzymes is facilitated by the crystal struc-tures in several reports, specifically: human KAT I in complexwith phenylalanine or indole-3-acetate [24,25]; human KAT II incomplex with kynurenine [28]; mouse KAT III structure in com-plex with kynurenine [23]; and mouse mAspAT in complex withkynurenine (present study). The substrate α-carboxylate moietyforms a salt bridge with a structurally conserved Arg and formsa hydrogen bond with a structurally conserved Asn (Figure 5,the residue with black background). The presence of this Arg

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Mouse mitochondrial aspartate aminotransferase/kynurenine aminotransferase-IV

Figure 5 Comparison of the residues implicated in substrate binding of four selected KATsThe Arg and Asn residues which bind the substrate α-carboxylate moiety are indicated with white lettering on blackbackground.

residue is a strictly conserved hallmark of all those membersof the aminotransferase superfamily whose structures have beendetermined to date [27]. The recognition of the substrate sidechain is achieved specifically by different structural determinantsin different KATs. The remarkable structural traits involved insubstrate side chain binding of human KAT I, human KAT II andmouse KAT III have been reviewed previously [14]. KAT Iand III have an aromatic hydrophobic pocket, which is largelyabsent in the mouse mAspAT structure. In contrast, the active siteof mAspAT tends to resemble KAT II, which may explain whymAspAT and KAT II have similar co-substrate specificity [29].

The identity of AspAT with KAT was first reported inE. coli [48] and when later mAspAT in mice, rats and humanswas found to have KAT activity, it was named KAT IV [22].It has been shown that mouse mAspAT has high transamina-tion activity towards glutamate and aspartate, and has detect-able activity towards phenylalanine, tyrosine, cysteine, trypto-phan, 3-HK, methionine, kynurenine and asparagine [33]. Inthis study, we demonstrated that mouse mAspAT could use α-oxoglutarate, phenylpyruvate, αKMB (α-oxo-γ -methiobutyricacid), indo-3-pyruvate, hydroxyphenylpyruvate, mercaptopyr-uvate, α-oxocaproic acid, oxaloacetate, α-oxobutyrate, pyruvateand glyoxylate as amino group acceptors. Mouse mAspAT wasfound to be a major player for the formation of KYNA in themouse brain [22], and this finding is supported by a recentstudy [33]. However, the inhibition study of mouse mAspATshows that aspartate, glutamate, glutamine, phenylalanine, tyr-osine, cysteine, tryptophan and histidine can competitively inhibitits KAT activity [33]. Since aspartate, glutamate and glutam-ine are the most abundant proteinogenic amino acids in mousebrains [49], and the specific KAT activity of mouse mAspAT isthe lowest of the four mKATs [33], the contribution of mousemAspAT to KYNA formation in mouse brains would be limitedunless kynurenine was highly sequestered from these abundantamino acids. Biochemically, mouse mAspAT primarily catalyses

the reversible transamination of oxaloacetate to aspartate in con-junction with the conversion of glutamate into α-oxoglutarate[50]. The enzyme has a number of specific roles in astrocytesand neurons in brains [51–55]. First, it has a role in the entry ofglutamate into the tricarboxylic acid cycle, and the re-synthesis ofintramitochondrial glutamate from tricarboxylic acid cycle inter-mediates [53,56–60]; secondly it has a key role in the synthesis ofthe neurotransmitter glutamate in brains [61,62]; and thirdly it isan essential component of the malate–aspartate shuttle, which isconsidered to be the most important mechanism for transferringreducing equivalents from the cytosol into the mitochondria inbrain tissue [55,58,63–66]. All of these functions of mAspAT arerelated to glutamate or aspartate. Therefore mAspAT may needto co-localize with these two amino acids, which may limit itsability to catalyse the formation of KYNA. Nevertheless, a con-siderable portion of the total KAT activity in mouse brain crudeproteins seems to be attributable to mAspAT [33], and moreoveran earlier report has also indicated that mAspAT was mainly re-sponsible for this KAT activity [22]. Accordingly, the specificcontribution of mAspAT in brain KYNA biosynthesis is yet to besubstantiated. A gene knock-out study in animal models couldaddress this question definitively.

In addition to the aforementioned roles played by mAspATas an aminotransferase, mAspAT is identical with a long-chainFABP (fatty acid-binding protein) [67–71]. In mammalian tis-sues, it was suggested that FABPs are involved in at least asubstantial portion of overall fatty acid uptake, the first step forthe involvement of fatty acids in cellular metabolism. In mice,KAT IV/mAspAT has the lowest KAT-specific activity amongthe four reported KATs [33], but it seems to be a primary con-tributor for the brain KAT activity in mouse brains [22,33]. Asquantity may compensate for low efficiency, this may indicatea significantly high mAspAT protein level. This relative abund-ance of mAspAT may enhance its function in long-chain fattyacid binding. As targeting fatty acid oxidation has been proposed

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as a therapeutic strategy for treating insulin resistance and the rateof fatty acid oxidation is largely affected by fatty acid availab-ility and its uptake into cells, one potential approach to treatinginsulin resistance is to decrease fatty acid uptake into heart orskeletal muscle [72]. Inhibiting the ability of mAspAT to binda fatty acid could lead to the reduction of cellular fatty acid up-take. Studying the mechanism of long-chain fatty acid binding ofmAspAT will help elucidate the protein inhibition or inactivation.Although molecular-modelling studies of the crystal structure ofmAspAT suggest that the identified pocket within the larger do-main of the enzyme might accommodate the typical long-chainfatty acid [73], whether this pocket serves as a fatty acid-bindingsite remains to be elucidated. A future study of co-crystallizationof mouse mAspAT and a long-chain fatty acid could identify thebinding site and provide the basis for investigating the inhibitionof the fatty acid binding.

AUTHOR CONTRIBUTION

Qian Han participated in the design of the study, carried out the ex-periments, performed analysis and wrote the manuscript. HowardRobinson carried out the experiments and performed analysis. TaoCai participated in the design of the study and helped to draft themanuscript. Danilo Tagle participated in the design of the studyand helped to draft the manuscript. Jianyong Li participated in thedesign of the study, carried out the experiments and wrote themanuscript.

ACKNOWLEDGEMENTS

We acknowledge support from the Virginia Tech Department ofBiological Sciences for the use of their X-ray facility and aregrateful to Dr Nancy Vogelaar for help with screening the crystalsprior to synchrotron data collection, to Haizhen Ding for help withprotein expression and to Elizabeth Watson for critically reading thispaper.

FUNDING

This work was supported by a grant from NINDS [NS062836] andby Intramural Research Programs of NIDCR and NINDS at NationalInstitutes of Health. The present study was carried out in partat the National Synchrotron Light Source, Brookhaven NationalLaboratory.

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Received 18 October 2010; accepted 26 October 2010Published as Immediate Publication 26 October 2010, doi 10.1042/BSR20100117

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