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Originally published In Press as doi:10.1074/jbc.M707925200 on December 7, 2007 Originally published In Press as doi:10.1074/jbc.M707925200 on December 5, 2007

J. Biol. Chem., Vol. 283, Issue 6, 3559-3566, February 8, 2008
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Crystal Structure of Human Kynurenine Aminotransferase II, a Drug Target for the Treatment of Schizophrenia*

Franca Rossi{ddagger}1, Silvia Garavaglia{ddagger}1, Valeria Montalbano{ddagger}, Martin A. Walsh§, and Menico Rizzi{ddagger}2

From the {ddagger}DiSCAFF-INFM, University of Piemonte Orientale "A. Avogadro", 28100 Novara, Italy and §Medical Research Council France, c/o European Synchrotron Radiation Facility, 38043 Grenoble Cedex, France

Received for publication, September 21, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Kynurenic acid is an endogenous neuroactive compound whose unbalancing is involved in the pathogenesis and progression of several neurological diseases. Kynurenic acid synthesis in the human brain is sustained by the catalytic activity of two kynurenine aminotransferases, hKAT I and hKAT II. A wealth of pharmacological data highlight hKAT II as a sensible target for the treatment of neuropathological conditions characterized by a kynurenic acid excess, such as schizophrenia and cognitive impairment. We have solved the structure of human KAT II by means of the single-wavelength anomalous dispersion method at 2.3-Å resolution. Although closely resembling the classical aminotransferase fold, the hKAT II architecture displays unique features. Structural comparison with a prototypical aspartate aminotransferase reveals a novel antiparallel strand-loop-strand motif that forms an unprecedented intersubunit β-sheet in the functional hKAT II dimer. Moreover, the N-terminal regions of hKAT II and aspartate aminotransferase appear to have converged to highly similar although 2-fold symmetry-related conformations, which fulfill the same functional role. A detailed structural comparison of hKAT I and hKAT II reveals a larger and more aliphatic character to the active site of hKAT II due to the absence of the aromatic cage involved in ligand binding in hKAT I. The observed structural differences could be exploited for the rational design of highly selective hKAT II inhibitors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Kynurenic acid (KYNA)3 is one of the neuroactive metabolites of the kynurenine pathway, the main route of oxidative tryptophan degradation in most living organisms (1). At concentrations recorded in the mammalian brain, KYNA antagonizes both the {alpha}7 nicotinic acetylcholine receptor ({alpha}7-nAChR) and the glycine co-agonist site of N-methyl-D-aspartate (NMDA) receptor, suggesting possible functions in brain physiology (2-5). Notably, given the critical role played by {alpha}7-nAChR and NMDA receptors in the brain, abnormal KYNA disposition may contribute to the pathogenesis and progression of neurological or psychiatric diseases that are associated with impaired cholinergic and/or glutamatergic neurotransmission (6). Indeed, reductions in endogenous brain KYNA lead to augmented neuronal vulnerability to NMDA receptor-mediated excitotoxic insults (7), whereas pharmacologically induced increases in KYNA provide neuronal protection against ischemic damage and have anticonvulsant effects (8, 9). Neurochemical studies show that KYNA-induced inhibition of {alpha}7-nAChRs causes a reduction in glutamate release and, secondarily, a decrease in extracellular dopamine levels (10). Inhibition of KYNA formation, on the other hand, results in an elevation in striatal dopamine levels, indicating a bi-directional modulation of dopaminergic neurotransmission by KYNA (11, 12). Taken together, these and other supportive data from animals and humans (13-16) suggest that KYNA may play a pathophysiologically significant role in the onset and progression of catastrophic brain diseases that are linked to a dysfunction of classic neurotransmitter systems.

In the mammalian central nervous system (CNS), KYNA is synthesized by the irreversible transamination of the non-standard amino acid L-kynurenine (L-KYN) (Fig. 1), which is produced by the first two catalyzed steps of the kynurenine pathway (1, 17). Two kynurenine aminotransferase isozymes, termed KAT I (identical to glutamine transaminase K, EC 2.6.1.6 [EC] 4) and KAT II (corresponding to {alpha}-aminoadipate aminotransferase, EC 2.6.1.7 [EC] ), are believed to quantitatively satisfy KYNA requirement in human, rat, and mouse brain (18-21). In the CNS, both isozymes are expressed preferentially in astrocytes (22, 23) and are capable of catalyzing the transamination of L-KYN to KYNA, although substantially differing in their biochemical properties (24, 25). Very recently, the mitochondrial aspartate aminotransferase from rat and human brain has also been shown to be able to transaminate L-KYN (26) and a third putative KAT-coding sequence has been identified in mouse (27); however, their role in the synthesis of cerebral KYNA in vivo remains to be established. Because of the reported catalytic promiscuity of KAT I (28) and the inhibition of the KAT I-dependent L-KYN transamination by physiological concentrations of glutamine and other amino acids (25), KAT II is currently viewed as the major determinant of KYNA biosynthesis in the human brain (29). KATs belong to the {alpha}-family of pyridoxal-5'-phosphate (PLP)-dependent enzymes (30) and have been assigned to aminotransferase subfamily I (AT I subfamily) on the basis of structural comparison and/or advanced phylogenetic analyses (31-33). To date, the structure of human KAT I (34) together with a few putative KAT II orthologs from bacteria and arthropods (35, 36) have been determined, but no structural data are known for human KAT II.


Figure 1
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  		FIGURE 1.
Scheme of the transamination reaction catalyzed by kynurenine aminotransferases. The synthesis of kynurenic acid is fully irreversible.

 
Although these studies provided an important contribution toward the understanding of the catalysis carried out by KATs, neither the KAT II active site nor the molecular determinants controlling the different enzymatic properties displayed by KAT I and KAT II have been described. In the present work, we report the crystal structure of recombinant human KAT II in its PLP form at 2.3-Å resolution. Our results complete the structural description of enzymes currently known to participate in KYNA biosynthesis in human brain and allow a direct structural comparison between KAT II and KAT I. Our observations led to the identification of the structural traits responsible for substrate recognition in KAT II and of the molecular determinants that fine tune catalysis in KAT I and KAT II.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Enzyme Expression and Purification
Construction of Expression Vector for hKAT II—Total RNA was extracted from a human neuroblastoma cell line (CRL-2266TM) using the Tri Reagent (Sigma). Approximately 100 ng of total RNA were subjected to reverse transcription using an oligo-dT primer and the ImProm-IITM polymerase (Promega), following the manufacturer's instructions. The open reading frame coding for human KAT II (residues 2-425, GenBankTM Accession Number NP_872603 [GenBank] ), was isolated by amplification on the obtained cDNA mixture, using the Expand High-Fidelity PCR System (Roche Applied Science) and the oligonucleotides hKAT II_for (5'-GACGCTCGAGAATTACGCACGGTTCATCACG, XhoI-site underlined) and hKAT II_rev (5'-ATACGGATCCTCATAAAGATTCTTTTATAAGTTGT, BamHI-site underlined) as the PCR primers pair. The BamHI-XhoI double-digested PCR product was ligated into the pET16b expression vector (Novagen), linearized by the same restriction enzymes. The DNA fragment coding for the His-tagged hKAT II in the resulting pET-hKAT II construct was fully sequenced.

Expression of Native hKAT II and SeMet-hKAT II—pET-hKAT II was transformed either in BL21(DE3) or in methionine-auxotrophic B834(DE3) competent bacteria for the expression of native and selenomethionine (SeM)-incorporated enzyme, respectively. pET-hKAT II-transformed BL21(DE3) bacteria were inoculated in 1 liter of LB medium (50 µg/ml ampicillin) whereas the pET-hKAT II-transformed B834(DE3) strain was inoculated in ampicillin-selective minimal M9 medium (37), supplemented by all amino acids at 40 µM concentration with the exception of methionine, that was added at 60 µM concentration in the form of a 75:25 mixture of SeM:methionine. Both bacterial cultures were grown at 22 °C under vigorous shaking, omitting the addition of isopropyl-1-thio-β-D-galactopyranoside. 18-20-h post-inoculation, bacteria were collected by centrifugation (11,000xg for 15 min at 4 °C), washed once in phosphate-buffered saline and flash-frozen in liquid nitrogen for storage at -20 °C until subsequent use.

Ni-NTA-based Purification of Native hKAT II and SeM-hKAT II—Bacterial pellet from 1 liter of native hKAT II expressing culture was dissolved in 40 ml of buffer A (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 40 µM PLP and a commercial proteases inhibitors mixture (Sigma)) and disrupted by sonication. The clarified bacterial lysate was added with 50 mM imidazole and incubated with 5 ml of drained Ni-NTA-agarose resin (Qiagen) for 1 h at 20 °C, under gentle agitation. After extensive washes in buffer A containing 75 mM imidazole, the elution of the absorbed protein was obtained at 200 mM imidazole in buffer A. Pure hKAT II containing fractions, as determined by standard SDS-PAGE analysis, were pooled, dialyzed against buffer A, and concentrated using a 30,000 MWCO disposable device (Vivascience) to a final protein concentration of 16 mg/ml, as determined by a Bradford assay (Sigma), using bovine serum albumin as the standard. We adopted the same procedure to purify and concentrate to 4.5 mg/ml the SeM-hKAT II enzyme, adding 40 µM dithiothreitol at each purification step. Aliquots of both hKAT II and SeM-hKAT II protein solutions were stored at 4 °C (up to 2 weeks storage) or at -80 °C (long term storage).

Crystallographic Study
Crystallization—The recombinant protein used in the crystallization experiments was proven to be active on the physiological substrate L-KYN.4 Initial crystallization conditions were identified by means of a hanging drop-based spare-matrix strategy by using the crystal screen kits from Hampton Research. The best crystals were obtained by mixing 1 µl of a protein solution at 16 mg/ml with an equal volume of a reservoir solution containing 22% p/v polyethylene glycol 3350, 0.3 M potassium iodide, 0.1 M Tris-HCl, pH 8.5 and equilibrating the resulting drop against 500 µl of the reservoir solution, at 20 °C. Needle-shaped yellow crystals grew to a maximum dimension of 0.7 x 0.2 x 0.05 mm in about 3 weeks. Crystals of SeM-hKAT II were obtained under the same conditions described above for native enzyme.

Structure Determination—For x-ray data collection, crystals were quickly equilibrated in a solution containing the crystallization buffer and 15% glycerol as the cryo-protectant and flash-frozen at 100 K under a stream of liquid nitrogen. Data to 2.3-Å resolution were collected from a native crystal of hKAT II at beamline ID23-1 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Analysis of the diffraction data set collected, allowed us to assign the crystal to the orthorhombic space group P21212, with cell dimensions a = 99.63 Å, b = 154.95 Å, and c = 61.20 Å, containing two molecules per asymmetric unit with a corresponding solvent content of 50%. The native data were processed using the programs MOSFLM (38) and the CCP4 suite of programs (39) were used for scaling. The structure of hKATII was solved by the single-wavelength anomalous dispersion (SAD) method from a selenomethionine-labeled crystal using data collected at the UK MAD beamline BM14 at the ESRF. The SAD data were collected from a randomly oriented crystal at the Se absorption edge peak based on an x-ray fluorescence scan acquired from the crystal at the beamline before data collection. Selenium sites were found by using SHELXD (40) and phasing was performed with SHARP (41). The resulting electron density map was of high quality and allowed the automatic tracing by the program ARP/wARP (42) that was also used for adding solvent molecules. The program O (43) was used for manual rebuilding and the program Refmac5 (44) for the crystallographic refinement. Data collection, phasing, and refinement statistics are given in Table 1.


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  		TABLE 1
Data collection, phasing, and refinement statistics

 


Figure 2
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  		FIGURE 2.
Stereo ribbon representation of the hKAT II subunit. The N-terminal motif and the large domain are colored in magenta; the small domain is colored in blue. The enzyme active site can be observed at the domain interface where the PLP cofactor molecule, in its internal aldimine form, is shown as ball-and-stick. The N and C termini of the molecule, the S2/S3 motif and the domain-connecting H13 {alpha}-helix are indicated. The secondary structural elements of the N-terminal motif as well as residues delimiting the disordered region are indicated. The figure was generated by BOBSCRIPT (47).

 
Deposition—The atomic coordinates and structure factors of human KAT II in its PLP form have been deposited with the Protein Data Bank with the accession code 2vgz and r2vgzsf.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Quality of the Model—The three-dimensional structure of the recombinant human kynurenine aminotransferase II in its PLP form (hKAT II) was solved by means of the SAD technique and refined at a resolution of 2.3 Å. A homodimer is present in the asymmetric unit with subunits related by a local dyad axis. Each subunit of the refined model contains 411 of 425 residues and one PLP molecule. None or poor electron density was visible for the exogenous N-terminal tag region and for residues Ile-19 to Lys-31 that have not been included in the final hKAT II model. The stereochemistry of the refined model has been assessed with the program PROCHECK (45). 92% of the residues were in the most favored regions of the Ramachandran plot and Pro-140 and Pro-203 were recognized as cis residues. Although Leu-293 falls in a disallowed region of the Ramachandran plot, the excellent electron density allowed us to unambiguously assign the observed conformation. Throughout the text, residues belonging to the symmetry related subunit in the functional hKAT II dimer are marked with an asterisk. All figures have been generated with the programs MOLSCRIPT (46), BOBSCRIPT (47) and PYMOL (48) as indicated in the figure legends.

hKAT II Overall Structure—While largely resembling the classical fold featuring aminotransferases (49, 50), the observed hKAT II molecular architecture displays degrees of novelty (Fig. 2). Each hKAT II subunit consists of an N-terminal motif (residues 13-46), a large domain (residues 47-326) and a small domain (residues 327-425). The N-terminal motif, budding from the body of the molecule, consists of two short {alpha}-helices at its ends, an internal β-strand and connecting coils (Fig. 2). Although its central region (residues 19-31) cannot be structurally assigned because of its high disorder, to our best knowledge, the topology observed for the hKAT II N-terminal motif has never been reported in the structure of any aminotransferase. A unique strand/loop/strand motif (S2/S3) marks the boundary between the N-terminal element and the large domain. The latter is organized around a canonical PLP-binding inner core whose topology, consisting of a mainly parallel seven-stranded β-sheet sandwiched between {alpha}-helices, is strictly conserved throughout PLP-dependent aminotransferases (30). A 42-residue-long, sharply bent {alpha}-helix (H13 in Fig. 2), leads into the small domain, that folds into a four-stranded antiparallel β-sheet shielded from the bulk solvent by further four {alpha}-helices. In contrast to what observed in the majority of aminotransferases, only residues of the C-terminal portion of the enzyme build up the small domain with no contribution from the N-terminal region. In each subunit, the main inter-domain contacts are provided by residues belonging to regions 351-354 and 387-390 of the small domain and residues standing at the inner edge of the large domain β-sheet. Interestingly, a salt bridge (distance 4.1 Å) is established between Lys-59, belonging to the unique S2/S3 motif, and Glu-309, standing at the 90 ° kink of the L-shaped {alpha}-helix connecting the large and the small domains. Such an interaction could play a role in maintaining the observed reciprocal orientation of the domains in each subunit.


Figure 3
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  		FIGURE 3.
Ribbon representation of the functional dimer of hKAT II. The two subunits are differently colored, and the PLP molecules are shown as ball-and-stick. The structure is viewed along the non-crystallographic dyad axis passing through the center of the unprecedented four stranded β-sheet built up by the S2/S3 motifs of the two subunits. The figure was generated by MOLSCRIPT (46).

 


Figure 4
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  		FIGURE 4.
Ribbon representation of the hKAT II (magenta) and T. thermophilus HB8 aspartate aminotransferase (blue) monomers after optimal superimposition. The N and C termini and the N-terminal regions of the two enzymes are indicated. The unique hKAT II S2/S3 motif and the T. thermophilus aspartate aminotransferase structurally equivalent H2 {alpha}-helix, are shown. The dyad axis relating the subunits in the functional dimers of both enzymes is indicated by a dotted line. The N-terminal regions of the two enzymes can be superimposed upon a 180 ° rotation around the indicated dyad axis. The PLP cofactors are drawn as ball-and-stick. The figure was generated by MOLSCRIPT (46).

 
hKAT II crystallized as an intimate dimer, with its two active sites hosted at the inter-domain interface in each subunit and at the intersubunit interface in the dimer (Fig. 3). Several regions contribute to the overall stability of the functional hKAT II dimer; among other, main intersubunit contacts are provided by the s1 β-strand and the h2 {alpha}-helix of the N-terminal motif and by the S14 β-strand of the opposite subunit small domain. A striking hKAT II structural element, critically contributing to the overall stability of the functional dimer, is represented by a four-stranded antiparallel flat β-sheet, formed by the unique S2/S3 motifs, that appears to sealing the two large domains together. Such an intersubunit β-sheet has never been observed in the structure of any aminotransferase reported so far, therefore representing an element of novelty.

Interestingly, an anion binding site, located at the intersubunit interface, was observed in the structure of hKAT II functional dimer. Indeed, inspection of the difference Fourier map calculated on the refined model, revealed the presence of a strong peak (15 sigma above the density r.m.s.d.) at a distance of 3.5 Å from residue Lys-123 of both subunits. We interpreted this peak to be an iodide ion due to the presence of 0.3 M potassium iodide in the crystallization buffer; however, the observed pocket would also allow the binding of a different anion, suggesting a possible contribution toward hKAT II dimer stability.

Structural Comparison with Related PLP Enzymes—A DALI-based search (51) was performed to identify hKAT II structural homologs present in the Protein Data Bank. The search revealed a significant structural homology with several aminotransferases but, to our surprise, not to the structure of the putative hKAT II ortholog from Pyrococcus horikoshii (35). The following two structures produced the DALI highest scores: (i) an aminotransferase of unknown function from Thermotoga maritima (PDB code 1VP4, z-score: 37.6, sequence identity of 27%, r.m.s.d. of 2.0 Å for 326 C{alpha} pairs), and (ii) the aspartate aminotransferase from Thermus thermophilus HB8, in its unliganded open form (PDB code 1BJW [PDB] , z-score: 35.1, sequence identity of 23%, r.m.s.d. of 2.2 Å for 324 C{alpha} pairs) (52). A detailed structural comparison was conducted between hKAT II and T. thermophilus aspartate aminotransferase (hereafter reported as tAAT), taken as representative of the extensively characterized family of aminotransferases (49).

As expected, the highly structurally related regions in hKAT II and tAAT encompass the inner core of the large domains and the C-terminal region of the small domains (Fig. 4), while significantly different conformations can be observed elsewhere in the molecules. As anticipated, an element of structural novelty displayed by hKAT II is represented by its S2/S3 motif, topologically corresponding to the H2 {alpha}-helix in tAAT (Fig. 4). This exclusive feature of the hKAT II architecture plays the same structural role of the equivalent region in tAAT, i.e. a major contribution to dimer stability. A further remarkable structural difference between the two enzymes is observed for their N-terminal regions that appear structurally unrelated in the optimally superimposed structures (Fig. 4). However, these regions become in fact superimposable (r.m.s.d. of 1.4 Å based on 15 C{alpha} pairs) when a 180 ° rotation around the hKAT II local 2-fold axis is applied, revealing a striking symmetry relationship. In the hKAT II dimer, the N-terminal region of each subunit contributes to build up the active site of the symmetry mate (Fig. 3), whereas in tAAT, the structurally equivalent region is involved in shaping the active site of its own subunit (52). Therefore, the N-terminal regions of hKAT II and tAAT appear to have converged to a highly similar architecture required for sustaining the same functional role (structuring the enzyme active site), although with pseudo 2-fold symmetry related conformations. The conformation shown by the N-terminal region in hKAT II is not unprecedented among PLP-dependent enzymes and resembles what observed in the N-terminal subdomain of members of PLP-dependent lyases (49), such as the cystathionine β-lyase from Escherichia coli (53). Indeed, this β-lyase was identified as a far structural ortholog of hKAT II in our DALI-based search (PDB code 1CL2, z-score: 19.4, sequence identity of 12%, r.m.s.d. of 3.9 Å for 281 C{alpha} pairs). Interestingly, hKAT II contains a structural element found in members of a functionally and structurally distinct PLP-dependent enzymes subfamily.


Figure 5
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  		FIGURE 5.
Stereo view of the hKAT II active site with the PLP cofactor in its internal aldimine form. The protein residues and the cofactor are depicted as ball-and-stick. Portion of the 2Fo-Fc electron density map, covering the PLP cofactor covalently bound to the catalytic residue Lys-263, is shown contoured at 1 sigma level. The figure was generated by BOBSCRIPT (47).

 
The PLP Binding Site—Each of the two active sites present in hKAT II functional dimer, sits in a wide and deep cleft built up by residues contributed by both domains of one subunit and by regions of the N-terminal motif and the large domain of the opposite one. Within each active site, the PLP-binding pocket harbors the cofactor molecule in Schiff-base linkage (C4'= N) to the {epsilon}-amino group of the catalytic residues Lys-263 of the correspondent subunit. The bottom of the PLP binding pocket is defined exclusively by residues pointing toward the PLP ring from the inner edge of the large domain β-sheet of the correspondent monomer. The active site wall on the pyridine side of the PLP cofactor is defined by residues provided by both the small and large domains of the correspondent subunit, while stretches 38-42 and 73-76 of the adjacent subunit form the active site wall facing the internal aldimine on the Schiff-base side. Residues of the large domains of both monomers, surrounding the PLP phosphate moiety, build up the remaining portion of the catalytic site. Several residues are in direct contact with the PLP molecule and participate to fix the observed orientation (Fig. 5). In particular, the nitrogen atom in the PLP pyridine ring is in contact with the extremely conserved residue Asp-230 (PLP N1, 3.6 Å, O{delta}2 Asp-230), whose side chain is held in place by an hydrogen bond established between its O{delta}1 position and the Tyr-195 OH group (distance: 2.7 Å); the cofactor O3' atom is tightly hydrogen bonded to the OH group of Tyr-233 (distance: 2.6 Å), that points toward the PLP molecule from the bottom of the active site, and it is also in contact with Asn-202 N{delta}2 (PLP O3', 3.2 Å, N{delta}2 Asn-202). The resulting bonding network strongly affects the PLP plane orientation (C4-C4'= N angle: 108.0 °), resulting in the absence of a perfect {pi}-stacking of its pyridine ring to the side chain of Tyr-142, which is placed just above the cofactor molecule. To the best of our knowledge, the presence of a tyrosine in this position is uncommon in aminotransferases, where tryptophan or phenylalanine is the most frequently observed residue. Interestingly, a structurally equivalent tyrosine residue characterizes instead a number of PLP enzymes involved in trans-sulfuration (e.g. cystathionine {gamma}-lyases and cystathionine β-lyases), where it has been suggested to fulfill different functions depending on the catalyzed chemical reaction type (53, 54). Therefore, as already underlined when describing the N-terminal motif, hKAT II reveals intriguing structural determinants in common with PLP-dependent lyases. We are therefore tempted to speculate that hKAT II could exhibit, beside its kynurenine aminotransferase activity, a still unknown biochemical function. An extensive characterization of the recombinant enzyme will be undertaken to assess this hypothesis.

The PLP phosphate group establishes a network of strong contacts with surrounding enzyme residues, which are highly conserved throughout PLP-dependent enzymes (55). In particular, the OP1 oxygen is involved in hydrogen bonding with the side chains of Ser-262 (PLP OP1-2.5Å-OH Ser-262) and Arg-270 (PLP OP1-2.5 Å - N{eta}2 Arg-270) of the same subunit and with the phenolic oxygen of Tyr-74 of the opposite monomer (PLP OP1-2.7 Å - OH Tyr-74*); the OP2 atom is in contact, at a mean distance of 3.0 Å, with the main-chain nitrogen atoms of Gln-118 and Ser-117; this residue also participates to the binding of the cofactor OP3 atom that is further engaged in a strong hydrogen bond with the side chain of Ser-260 (PLP OP3-2.5Å-OH Ser-260), whereas Gln-118 binds the OP4 at 2.9-Å distance from its N{epsilon}2 position. Finally, in each subunit, the positive macro-dipole of the {alpha}-helix H6, pointing to the cofactor molecule, compensates the negative charge of the PLP phosphate moiety.


Figure 6
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  		FIGURE 6.
Representation of the hKAT II and hKAT I active sites, after optimal superposition of the two structures. The residues involved in L-Phe binding in hKAT I together with their structurally equivalent residues in hKAT II are colored in white and green, respectively. The L-Phe sits above the PLP cofactor as observed in the structure of hKAT I:L-Phe complex (PDB code 1W7M) (34). The figure was generated by MOLSCRIPT (46).

 
Structural Comparison to Human KAT I and Substrate Modeling—The synthesis of KYNA in the human brain is sustained by the catalytic activity of the two aminotransferases hKAT I and hKAT II (see Introduction). Although the individual contribution of each of the two isozymes to the synthesis of KYNA still needs to be precisely quantified, hKAT II is currently the primary target for the development of pharmacological tools aimed at lowering KYNA concentration in the brain. However, such a therapeutic intervention needs to take into account the possible pro-neurotoxic effects resulting from an excessive reduction of brain KYNA concentration (6, 12). Therefore, the identification of the molecular determinants driving substrate recognition and catalysis in human kynurenine aminotransferases is of primary importance for the rational design of potent, specific hKAT II inhibitors.

Unfortunately, all attempts to solve the structure of hKAT II in complex with the physiological substrate L-KYN have so far been unsuccessful. This is most likely due to the catalytic competence of our crystals, as indicated by the fact that they became colorless upon L-KYN addition. This also caused a severe crystal suffering resulting in poor or no x-ray diffraction. Therefore, the L-KYN binding mode in hKAT II will be described based on a model prepared by optimal superimposition onto the experimentally determined structure of hKAT I in complex with the competitive inhibitor L-phenylalanine (34, 25). hKAT I and hKAT II share a 15% overall sequence identity and their structures can be superposed with a r.m.s.d. of 3.0 Å based on 245 C{alpha} pairs. Although to a sensibly less extent, the structural homology with hKAT I covers the same regions described when comparing hKAT II with tAAT. Indeed, the structural homology between the two human isozymes is restricted to the inner core of the large domain and to the C-terminal region of the small domain, while severe deviations are observed for the rest of the molecule, including the unique hKAT II S2/S3 motif and N-terminal region. Inspection of the active sites of hKAT I and hKAT II revealed a striking difference in their substrate binding pockets (Fig. 6A). Indeed, the major molecular determinant responsible for substrate recognition in hKAT I, namely an aromatic cage built up by residues Tyr-63*, His-279*, Phe-278*, Tyr-101, Phe-125, and Trp-18, is largely absent in the structure of hKAT II where the equivalent positions are occupied by polar or neutral amino acids. Of particular relevance is the absence in hKAT II of the structurally equivalent position occupied by Trp-18 in hKAT I; this residue was indeed shown to play a major role in substrate recognition, being recruited at the active site upon L-Phe binding (34). Overall, our structural data reveal a less aromatic environment in the active site of hKAT II as compared with hKAT I; in addition, the substrate-binding pocket in hKAT II appears sensibly wider, mainly due to the above mentioned absence of a residue equivalent to hKAT I Trp-18 and to the substitution of hKAT I Phe-278* with Leu-293*. These structural features suggest a lower affinity of hKAT II toward L-KYN with respect to hKAT I, as indeed reported for the rat orthologous enzymes (24). Because we were unable to determine the three-dimensional structure of the Michaelis-Menten complex with the physiological substrate L-KYN, we propose here a model of L-KYN binding based on the structure superimposition of hKAT II to hKAT I in complex with L-Phe. According to the resulting L-KYN binding mode (Fig. 7), two molecular determinants appear to be of particular relevance for substrate binding and recognition. Major contributions toward L-KYN binding are provided by a highly conserved arginine residue (Arg-399), that anchors the substrate {alpha}-carboxylic group through a salt bridge and by two tyrosine residues contacting the L-KYN side chain; as shown in Fig. 7, the benzyl group of Tyr-142 is properly oriented to establish an aromatic hydrogen bond with the anthranilic amino group of L-KYN that in turn is hydrogen-bonded to the hydroxyl group of the strictly conserved Tyr-74*. Based on our structural analysis, the different sterical requirements featuring hKAT I and hKAT II active site could be exploited for the rational design of highly specific hKAT II inhibitors. Indeed, while in the hKAT I:L-KYN complex the substrate anthranilic moiety perfectly fits the narrow and highly aromatic ligand binding site, some empty space is left in the case of the hKAT II:L-KYN complex. We therefore propose the anthranilic moiety as a hot spot for the chemical modification of substrate-like highly selective hKAT II inhibitors. Increasing the steric hindrance of the anthranilic moiety would hamper binding to hKAT I, but would be tolerated by the hKAT II active site.

In conclusion, the determination of the three-dimensional structure of hKAT II may help the rational design of isozyme-specific inhibitors, aimed at a fine-tuned modulation of KYNA levels in neuropathological conditions including mental illness and cognitive impairment (13-16) without exposing the brain to an abrupt and total loss of KAT activity and to the consequent deleterious effects on CNS function by the resulting KYNA deprivation.


Figure 7
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  		FIGURE 7.
Modeling of the L-kynurenine binding in human KATs after optimal superimposition of the two structures. Close up view of the enzyme active site of hKAT I (A) and hKAT II (B) with modeled L-KYN. The reported substrate orientation in the enzyme active site was obtained by closely mimicking the conformation adopted by L-Phe in the three-dimensional structure of hKAT I:L-Phe complex (34). Residues suggested to providing major contribution toward ligand recognition are indicated. Schemes were generated with PYMOL (48).

 

   FOOTNOTES
 
The atomic coordinates and structure factors (code 2vgz) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by a grant from the Regione Piemonte (CIPE 2004) and by MUR (Interlink, 2004). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to the work. Back

2 To whom correspondence should be addressed: DiSCAFF, University of Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy. Tel.: 39-0321-375712; Fax: 39-0321-375821; E-mail: rizzi{at}pharm.unipmn.it.

3 The abbreviations used are: KYNA, kynurenic acid; CNS, central nervous system; PLP, pyridoxal-5'-phosphate; NTA, nitrilotriacetic acid; r.m.s.d., root mean square deviation; PDB, Protein Data Bank. Back

4 F. Rossi, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Robert Schwarcz from the Maryland Psychiatric Research Center (University of Maryland) for critical reading of the manuscript and helpful discussion. We thank the European Synchrotron Radiation Facility (ESRF, Grenoble, France) for data collections at the beam lines ID23-1 and BM14.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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Q. Han, H. Robinson, T. Cai, D. A. Tagle, and J. Li
Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III
Mol. Cell. Biol., February 1, 2009; 29(3): 784 - 793.
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