Portal de Periódicos da CAPES

Crystal Structure of Human Kynurenine Aminotransferase II

2007; Elsevier BV; Volume: 283; Issue: 6 Linguagem: Inglês

10.1074/jbc.m708358200

1083-351X

Qian Han, Howard Robinson, Jianyong Li,

Bipolar Disorder and Treatment

Human kynurenine aminotransferase II (hKAT-II) efficiently catalyzes the transamination of knunrenine to kynurenic acid (KYNA). KYNA is the only known endogenous antagonist of N-methyl-d-aspartate (NMDA) receptors and is also an antagonist of 7-nicotinic acetylcholine receptors. Abnormal concentrations of brain KYNA have been implicated in the pathogenesis and development of several neurological and psychiatric diseases in humans. Consequently, enzymes involved in the production of brain KYNA have been considered potential regulatory targets. In this article, we report a 2.16Å crystal structure of hKAT-II and a 1.95Å structure of its complex with kynurenine. The protein architecture of hKAT-II reveals that it belongs to the fold-type I pyridoxal 5-phosphate (PLP)-dependent enzymes. In comparison with all subclasses of fold-type I-PLP-dependent enzymes, we propose that hKAT-II represents a novel subclass in the fold-type I enzymes because of the unique folding of its first 65 N-terminal residues. This study provides a molecular basis for future effort in maintaining physiological concentrations of KYNA through molecular and biochemical regulation of hKAT-II. Human kynurenine aminotransferase II (hKAT-II) efficiently catalyzes the transamination of knunrenine to kynurenic acid (KYNA). KYNA is the only known endogenous antagonist of N-methyl-d-aspartate (NMDA) receptors and is also an antagonist of 7-nicotinic acetylcholine receptors. Abnormal concentrations of brain KYNA have been implicated in the pathogenesis and development of several neurological and psychiatric diseases in humans. Consequently, enzymes involved in the production of brain KYNA have been considered potential regulatory targets. In this article, we report a 2.16Å crystal structure of hKAT-II and a 1.95Å structure of its complex with kynurenine. The protein architecture of hKAT-II reveals that it belongs to the fold-type I pyridoxal 5-phosphate (PLP)-dependent enzymes. In comparison with all subclasses of fold-type I-PLP-dependent enzymes, we propose that hKAT-II represents a novel subclass in the fold-type I enzymes because of the unique folding of its first 65 N-terminal residues. This study provides a molecular basis for future effort in maintaining physiological concentrations of KYNA through molecular and biochemical regulation of hKAT-II. Kynurenic acid (KYNA) 2The abbreviations used are: KYNAkynurenic acidhKAT-IIhuman kynurenine aminotransferase IIKATkynurenine aminotransferaseLLPlysine-pyridoxal-5′-phosphatePLPpyridoxal-5′-phosphatePMPpyridoximine 5′-phosphater.m.s.root mean squareKYNkynurenine. is the only known endogenous antagonist of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors (1Leeson P.D. Iversen L.L. J. Med. Chem. 1994; 37: 4053-4067Crossref PubMed Scopus (317) Google Scholar, 2Perkins M.N. Stone T.W. Brain Res. 1982; 247: 184-187Crossref PubMed Scopus (699) Google Scholar, 3Stone T.W. Perkins M.N. Neurosci. Lett. 1984; 52: 335-340Crossref PubMed Scopus (48) Google Scholar, 4Birch P.J. Grossman C.J. Hayes A.G. Eur. J. Pharmacol. 1988; 154: 85-87Crossref PubMed Scopus (273) Google Scholar). KYNA is also the antagonist of α7-nicotinic acetylcholine receptor (5Pereira E.F. Hilmas C. Santos M.D. Alkondon M. Maelicke A. Albuquerque E.X. J. Neurobiol. 2002; 53: 479-500Crossref PubMed Scopus (179) Google Scholar, 6Hilmas C. Pereira E.F. Alkondon M. Rassoulpour A. Schwarcz R. Albuquerque E.X. J. Neurosci. 2001; 21: 7463-7473Crossref PubMed Google Scholar, 7Alkondon M. Pereira E.F. Yu P. Arruda E.Z. Almeida L.E. Guidetti P. Fawcett W.P. Sapko M.T. Randall W.R. Schwarcz R. Tagle D.A. Albuquerque E.X. J. Neurosci. 2004; 24: 4635-4648Crossref PubMed Scopus (126) Google Scholar, 8Stone T.W. Eur. J. Neurosci. 2007; 25: 2656-2665Crossref PubMed Scopus (84) Google Scholar). In mammalian brain, glutamate is the major excitatory neurotransmitter and acts through both ligand-gated ion channels and G-protein-coupled receptors, which are collectively called glutamate receptors (9Palmada M. Centelles J.J. Front. Biosci. 1998; 3: d701-d718Crossref PubMed Google Scholar). Activation of these receptors is responsible for basal excitatory synaptic transmission and many forms of synaptic plasticity such as long-term potentiation and long-term depression, which are thought to underlie learning and memory (9Palmada M. Centelles J.J. Front. Biosci. 1998; 3: d701-d718Crossref PubMed Google Scholar, 10Martin S.J. Grimwood P.D. Morris R.G. Annu. Rev. Neurosci. 2000; 23: 649-711Crossref PubMed Scopus (2105) Google Scholar). However, any event or process leading to a sudden or chronic increase in the activity of glutamate receptors often induces the death of neurons (11Ogura A. Miyamoto M. Kudo Y. Exp. Brain Res. 1988; 73: 447-458Crossref PubMed Scopus (164) Google Scholar). Consequently, a mechanism capable of preventing glutamate receptors from being overly stimulated seems essential for maintaining the normal physiological condition of the brain (12Stone T.W. Addae J.I. Eur. J. Pharmacol. 2002; 447: 285-296Crossref PubMed Scopus (86) Google Scholar, 13Stone T.W. Darlington L.G. Nat. Rev. Drug. Discov. 2002; 1: 609-620Crossref PubMed Scopus (646) Google Scholar). Brain KYNA levels are abnormal in the progression of diseases such as Huntington's disease (14Beal M.F. Matson W.R. Swartz K.J. Gamache P.H. Bird E.D. J. Neurochem. 1990; 55: 1327-1339Crossref PubMed Scopus (275) Google Scholar, 15Guidetti P. Reddy P.H. Tagle D.A. Schwarcz R. Neurosci. Lett. 2000; 283: 233-235Crossref PubMed Scopus (89) Google Scholar, 16Yu P. Mosbrook D.M. Tagle D.A. Mamm. Genome. 1999; 10: 845-852Crossref PubMed Scopus (22) Google Scholar) and schizophrenia (17Schwarcz R. Rassoulpour A. Wu H.Q. Medoff D. Tamminga C.A. Roberts R.C. Biol. Psychiatry. 2001; 50: 521-530Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 18Erhardt S. Blennow K. Nordin C. Skogh E. Lindstrom L.H. Engberg G. Neurosci. Lett. 2001; 313: 96-98Crossref PubMed Scopus (376) Google Scholar, 19Erhardt S. Schwieler L. Nilsson L. Linderholm K. Engberg G. Physiol. Behav. 2007; 92: 203-209Crossref PubMed Scopus (121) Google Scholar), which suggest that variations in brain KYNA, acting as an endogenous modulator of glutamatergic and cholinergic neurotransmission, may be functionally significant. Recently, KYNA has been identified as an endogenous ligand for an orphan G-protein-coupled receptor, GPR35, which is predominantly expressed in immune cells (20Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). The tryptophan metabolic pathway is activated under inflammatory conditions, such as viral invasion, presence of bacterial lipopolysaccharides or interferon stimulation (21Mellor A.L. Munn D.H. Nat. Rev. Immunol. 2004; 4: 762-774Crossref PubMed Scopus (1875) Google Scholar, 22Taylor M.W. Feng G.S. Faseb J. 1991; 5: 2516-2522Crossref PubMed Scopus (938) Google Scholar). The activation of tryptophan metabolism causes a reduced plasma tryptophan level and an elevation in KYNA concentration (13Stone T.W. Darlington L.G. Nat. Rev. Drug. Discov. 2002; 1: 609-620Crossref PubMed Scopus (646) Google Scholar). Therefore, this receptor-ligand pair may play a role in immunological regulation. kynurenic acid human kynurenine aminotransferase II kynurenine aminotransferase lysine-pyridoxal-5′-phosphate pyridoxal-5′-phosphate pyridoximine 5′-phosphate root mean square kynurenine. KYNA is produced enzymatically by irreversible transamination of kynurenine, the primary catabolic product of tryptophan, and the newly formed KYNA is secreted into the extracellular milieu (23Speciale C. Wu H.Q. Gramsbergen J.B. Turski W.A. Ungerstedt U. Schwarcz R. Neurosci. Lett. 1990; 116: 198-203Crossref PubMed Scopus (55) Google Scholar, 24Turski W.A. Gramsbergen J.B. Traitler H. Schwarcz R. J. Neurochem. 1989; 52: 1629-1636Crossref PubMed Scopus (198) Google Scholar, 25Swartz K.J. During M.J. Freese A. Beal M.F. J. Neurosci. 1990; 10: 2965-2973Crossref PubMed Google Scholar). In mammals, four proteins arbitrarily named as kynurenine aminotransferase (KAT) I, II, III, and IV, have been considered to be involved in KYNA synthesis in the CNS (26Guidetti P. Okuno E. Schwarcz R. J. Neurosci. Res. 1997; 50: 457-465Crossref PubMed Scopus (209) Google Scholar, 27Schwarcz R. Pellicciari R. J. Pharmacol. Exp. Ther. 2002; 303: 1-10Crossref PubMed Scopus (476) Google Scholar, 28Han Q. Li J. Li J. Eur. J. Biochem. 2004; 271: 4804-4814Crossref PubMed Scopus (85) Google Scholar, 29Yu P. Li Z. Zhang L. Tagle D.A. Cai T. Gene (Amst.). 2006; 365: 111-118Crossref PubMed Scopus (87) Google Scholar, 30Guidetti P. Amori L. Sapko M.T. Okuno E. Schwarcz R. J. Neurochem. 2007; 102: 103-111Crossref PubMed Scopus (134) Google Scholar). These proteins are all pyridoxal 5-phosphate-(PLP)-dependent enzymes. KAT-II is considered the principal enzyme responsible for the synthesis of KYNA in the rat brain (26Guidetti P. Okuno E. Schwarcz R. J. Neurosci. Res. 1997; 50: 457-465Crossref PubMed Scopus (209) Google Scholar, 27Schwarcz R. Pellicciari R. J. Pharmacol. Exp. Ther. 2002; 303: 1-10Crossref PubMed Scopus (476) Google Scholar). Mice in which KAT II was removed by genomic manipulation showed a substantial decrease in brain KYNA levels and presented with phenotypic changes that were in line with this chemical deficit (7Alkondon M. Pereira E.F. Yu P. Arruda E.Z. Almeida L.E. Guidetti P. Fawcett W.P. Sapko M.T. Randall W.R. Schwarcz R. Tagle D.A. Albuquerque E.X. J. Neurosci. 2004; 24: 4635-4648Crossref PubMed Scopus (126) Google Scholar, 31Yu P. Di Prospero N.A. Sapko M.T. Cai T. Chen A. Melendez-Ferro M. Du F. Whetsell Jr., W.O. Guidetti P. Schwarcz R. Tagle D.A. Mol. Cell. Biol. 2004; 24: 6919-6930Crossref PubMed Scopus (71) Google Scholar, 32Sapko M.T. Guidetti P. Yu P. Tagle D.A. Pellicciari R. Schwarcz R. Exp. Neurol. 2006; 197: 31-40Crossref PubMed Scopus (114) Google Scholar). Pharmacological tools exist to manipulate the kynurenine pathway and, thus, change the brain levels of KYNA (27Schwarcz R. Pellicciari R. J. Pharmacol. Exp. Ther. 2002; 303: 1-10Crossref PubMed Scopus (476) Google Scholar, 33Pellicciari R. Natalini B. Costantino G. Mahmoud M.R. Mattoli L. Sadeghpour B.M. Moroni F. Chiarugi A. Carpenedo R. J. Med. Chem. 1994; 37: 647-655Crossref PubMed Scopus (133) Google Scholar). Also, some studies are looking for agents that specifically inhibit KAT activity and, thus, reduce brain KYNA (28Han Q. Li J. Li J. Eur. J. Biochem. 2004; 271: 4804-4814Crossref PubMed Scopus (85) Google Scholar, 34Csillik A. Knyihar E. Okuno E. Krisztin-Peva B. Csillik B. Vecsei L. Exp. Neurol. 2002; 177: 233-241Crossref PubMed Scopus (23) Google Scholar, 35Luchowski P. Luchowska E. Turski W.A. Urbanska E.M. Neurosci. Lett. 2002; 330: 49-52Crossref PubMed Scopus (50) Google Scholar, 36Pellicciari R. Rizzo R.C. Costantino G. Marinozzi M. Amori L. Guidetti P. Wu H.Q. Schwarcz R. Chem. Med. Chem. 2006; 1: 528-531Crossref Scopus (73) Google Scholar). The crystal structures of human KAT-I (37Rossi F. Han Q. Li J. Li J. Rizzi M. J. Biol. Chem. 2004; 279: 50214-50220Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and its homologue, glutamine-phenylpyruvate aminotransferase from Thermus thermophilus HB8 (38Goto M. Omi R. Miyahara I. Hosono A. Mizuguchi H. Hayashi H. Kagamiyama H. Hirotsu K. J. Biol. Chem. 2004; 279: 16518-16525Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), have been determined. But, it seems difficult to build a three-dimensional model for hKAT-II based on the KAT-I structures because of poor amino acid sequence identities between KAT-I and KAT-II (11% for human enzymes). The crystal structure of an hKAT-II homologue from Pyrococcus horikoshii has also been determined (39Chon H. Matsumura H. Koga Y. Takano K. Kanaya S. Proteins. 2005; 61: 685-688Crossref PubMed Scopus (17) Google Scholar); however, it is still hard to do structure/ligand-based drug design based upon this structure because it only shares a 28% percent amino acid sequence identity with hKAT-II. Based on our research, we can report the crystal structure of hKAT-II at a resolution of 2.16 Å in its PLP form as well as the structure in complex with its substrate kynurenine at a resolution of 1.95 Å. Determination of the three-dimensional structure of hKAT-II may contribute to the rational design of selective inhibitors that are of intense medical interest with respect to a number of pathological conditions in humans. Expression and Purification and Activity Assay of Recombinant hKAT-IIhKAT-II coding sequence was amplified from a human liver cDNA pool using a forward primer (5′-AAAACATATGAATTACGCACGGTTCAT-3′) and a reverse primer (AAAACTCGAGTCATAAAGATTCTTTTATAAGTTG-3′) containing the gene-specific sequence (hKAT II, NP_057312) and NdeI and Xho restriction sites, respectively. The amplified sequence was cloned into an Impact™-CN plasmid (New England Biolabs) for expression of a fusion protein containing a chitin-binding domain. Transformed Escherichia coli cells were cultured at 37 °C. After induction with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside, the cells were cultured at 15 °C for 24 h. Six liters of cells were harvested as the start materials for affinity purification. The soluble fusion proteins were applied to a column packed with chitin beads and subsequently hydrolyzed under reducing conditions. The affinity purification resulted in the isolation of hKAT II at around 80% purity. Further purification of the recombinant hKAT-II was achieved by DEAE-Sepharose, hydroxyapatite, and gel-filtration chromatography. The purified recombinant hKAT-II was concentrated to 10 mg ml-1 protein in 5 mm phosphate buffer (pH7.5) using a Centricon YM-30 concentrator (Millipore). The KAT activity assay was based upon previously described methods (40Han Q. Fang J. Li J. J. Biol. Chem. 2002; 277: 15781-15787Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Briefly, a reaction mixture of 50 ml, containing 10 mml-kynurenine, 5 mm ketoglutarate, 40 mm PLP, and 2 μg of protein sample, was prepared using 100 mm potassium phosphate buffer (pH 7.5). The mixture was incubated for 15 min at 45 °C, and the reaction was stopped by adding an equal volume of 0.8 m formic acid. Supernatant of the reaction mixture, obtained by centrifugation at 15,000 × g for 10 min at 4 °C, was analyzed by HPLC with UV detection at 330 nm for both kynurenine and KYNA. hKAT-II CrystallizationThe crystals were grown by hanging-drop vapor diffusion methods with the volume of reservoir solution at 500 μl and the drop volume at 2 μl, containing 1 μl of protein sample and 1 μl of reservoir solution. The optimized crystallization buffer consisted of 15% PEG 10,000 and 0.1 m Hepes at pH 7.5. The crystals of the enzyme-substrate complexes were obtained by soaking the crystals in 2.5 mm of kynurenine in the above crystallization buffer for 3 days. Data Collection and ProcessingIndividual hKAT-II crystals were cryogenized using 25% glycerol in the crystallization buffer as a cryo-protectant solution. Diffraction data of hKAT-II crystals were collected at the Brookhaven National Synchrotron Light Source beam line X29A (λ = 1.0908 Å). Data were collected using an ADSC Q315 CCD detector. All data were indexed and integrated using HKL-2000 software; scaling and merging of diffraction data were performed using the program SCALEPACK (41Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The parameters of the crystals and data collection are listed in Table 1.TABLE 1Data collection and refinement statisticsCrystal datahKAT-IIhKAT-II:KYNSpace GroupP21Unit Cella (Å)109.570.8b (Å)71.0109.4c (Å)121.1119.4β (°)101.194.7Data collectionX-ray sourceBNLaBrookhaven National Laboratory. - X29Wavelength (Å)1.0809Resolution (Å)bThe values in parentheses are for the highest resolution shell.2.16 (2.23-2.16)1.95 (2.02-1.95)Total number of reflections721,6101,415,391Number of unique reflections96,642132,070R-mergebThe values in parentheses are for the highest resolution shell.0.12 (0.42)0.09 (0.37)RedundancybThe values in parentheses are for the highest resolution shell.8.1 (4.9)11.5 (6.5)Completeness (%)bThe values in parentheses are for the highest resolution shell.92.2 (75.8)93.2 (62.1)Refinement statisticsR-work (%)24.219.4R-free (%)25.622.2R.m.s. bond lengths (Å)0.0220.018R.m.s. bond angles (°)1.9821.796No. of ligand or cofactor molecules4 LLP4 PMP7 GOLcGOL, glycerol.4 KYN6 GOLcGOL, glycerol.No. of water molecules6121341Average B overall (Å2)37.629.6a Brookhaven National Laboratory.b The values in parentheses are for the highest resolution shell.c GOL, glycerol. Open table in a new tab Structure DeterminationThe structures of hKAT-II were determined by the molecular replacement method using the published bacterial homologue structure (Protein Data Bank code 1×0m, 28% amino acid sequence identical) (39Chon H. Matsumura H. Koga Y. Takano K. Kanaya S. Proteins. 2005; 61: 685-688Crossref PubMed Scopus (17) Google Scholar). The programs Phaser (42McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1602) Google Scholar, 43Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1103) Google Scholar, 44Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1373-1382Crossref PubMed Scopus (789) Google Scholar) and Molrep (45Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) were employed to calculate both cross-rotation and translation of the model. The initial model was subjected to iterative cycles of crystallographic refinement with the Refmac 5.2 (46Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) and graphic sessions for model building using the program O (47Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. 1991; A47: 110-119Crossref Scopus (13014) Google Scholar). The cofactor and substrate molecules were modeled when the R factor dropped to a value of around 0.25 at full resolution for all hKAT-II structures, based upon both the 2Fo-Fc and Fo-Fc electron density maps. Solvent molecules were automatically added and refined with ARP/wARP (48Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. 1997; D53: 448-455Google Scholar) together with Refmac 5.2. Structure AnalysisSuperposition of structures was done using Lsqkab (49Kabsch W. Acta Crystallogr. 1976; A32: 922-923Crossref Scopus (2380) Google Scholar) in the CCP4 suite. Figures were generated using Pymol (50DeLano W.L. The PyMOL Molecular Graphics System. Delano Scientific, San Carlos, CA2002Google Scholar). Protein and substrate interaction also was analyzed using Pymol (50DeLano W.L. The PyMOL Molecular Graphics System. Delano Scientific, San Carlos, CA2002Google Scholar). Enzyme Purification and Activity AssayUsing the affinity purification method, DEAE-Sepharose, hydroxyapatite, and gel filtration chromatography, we purified hKAT-II recombinant protein. The purified hKAT-II showed high activity toward kynurenine and ketoglutarate (Fig. 1). The identity of rat KAT-II to aminoadipate aminotransferase was confirmed 30 years ago (51Tobes M.C. Mason M. J. Biol. Chem. 1977; 252: 4591-4599Abstract Full Text PDF PubMed Google Scholar), and the KAT-II gene was isolated by RT-PCR from rat kidney (52Buchli R. Alberati-Giani D. Malherbe P. Kohler C. Broger C. Cesura A.M. J. Biol. Chem. 1995; 270: 29330-29335Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) or EST assembly from mouse (16Yu P. Mosbrook D.M. Tagle D.A. Mamm. Genome. 1999; 10: 845-852Crossref PubMed Scopus (22) Google Scholar) and human (53Goh D.L. Patel A. Thomas G.H. Salomons G.S. Schor D.S. Jakobs C. Geraghty M.T. Mol. Genet. Metab. 2002; 76: 172-180Crossref PubMed Scopus (30) Google Scholar). Recombinant mouse KAT-II is active to both aminoadipate and kynurenine and prefers ketoglutarate as an amino group receptor (52Buchli R. Alberati-Giani D. Malherbe P. Kohler C. Broger C. Cesura A.M. J. Biol. Chem. 1995; 270: 29330-29335Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Recombinant hKAT-II has shown activity toward aminoadipate and ketoglutarate (53Goh D.L. Patel A. Thomas G.H. Salomons G.S. Schor D.S. Jakobs C. Geraghty M.T. Mol. Genet. Metab. 2002; 76: 172-180Crossref PubMed Scopus (30) Google Scholar), and together with our results, we can conclude that hKAT-II is also active to both kynurenine and aminoadipate. Overall StructureThe structures of hKAT-II were determined by molecular replacement and refined to 1.95-Å resolution for the hKAT-II:kynurenine complex and 2.30-Å resolution for the hKAT-II PLP form. Final models contain 4 × 425 amino acid residues each and yield a crystallographic R value of 19.4% and an Rfree value of 22.2% for the hKAT-II:kynurenine complex, and 24.2 and 25.6% for its PLP form with ideal geometry (Table 1). There are four protein molecules in an asymmetric unit, which form two biological homodimers. The residues of the four subunits in hKAT-II structures are numbered 1 (A) to 425 (A) for chain A, 1 (B) to 425 (B) for chain B, 1 (C) to 425 (C) for chain C, and 1 (D) to 425 (D) for chain D. To distinguish residues from the two subunits of a biological dimer, we labeled residues from opposite subunit with a star (*), such as Tyr-74*. The results of the refinement are summarized in Table 1. All residues in all four chains except for Leu-293 are in favorable regions of the Ramachandran plot as defined with PROCHECK (54Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Although Leu-293 (A), (B), (C), and (D) in all structures falls within a disallowed region of the Ramachandran plot of the solved structures, the excellent electron density allowed us to unambiguously assign the observed conformations. An overview of the dimeric structure model is shown in Fig. 2a. The structure has an aspartate aminotransferase large and small domain (Fig. 3). The large domain (residues 76-323) contains a seven-stranded β-sheet (b3-b9), and the small domain comprises the C-terminal part of the chain (residues 324-428), which folds into a 2-stranded β-sheet (b10, b11) covered with helices on one side. The residue Asp-230 interacts with the pyridine nitrogen of the cofactor (Figs. 4 and 5), which is structurally and functionally conserved within fold-type I of the PLP-dependent enzyme family, and indicates its importance for catalysis. Based upon the above features of the structure, hKAT-II is a fold-type I PLP-dependent enzyme (55Jansonius J.N. Curr. Opin. Struct. Biol. 1998; 8: 759-769Crossref PubMed Scopus (363) Google Scholar, 56Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (343) Google Scholar, 57Kack H. Sandmark J. Gibson K. Schneider G. Lindqvist Y. J. Mol. Biol. 1999; 291: 857-876Crossref PubMed Scopus (97) Google Scholar, 58Schneider G. Kack H. Lindqvist Y. Structure. 2000; 8: R1-R6Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 59Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1993; 214: 549-561Crossref PubMed Scopus (363) Google Scholar).FIGURE 3Schematic view of the subunit of hKAT-II. The secondary structure elements, β-strands are numbered from 1 to 11. Substrate (Kyn) and cofactor (PMP) are included in stick models. Three parts, large domain (green), small domain (blue), and N-terminal part (pink) are labeled.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4The hKAT-II active site. A stereo view of the active site in the hKAT-II structure. The protein portions building up the active site and contributed to by the two subunits of the functional homodimer are shown in a schematic representation and are colored in deep teal (subunit A) and violet (subunit B). The PLP cofactor and the protein residues within a 5-Å distance of PLP are shown. Only the 2Fo-Fc electron density map covering the LLP 263 is shown contoured at the 1 sigma.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Kynurenine binding site. Stereoview of the kynurenine binding site in the hKAT-II:kynurenine complex structure. The protein portions building up the binding site and contributed to by the two subunits of the functional homodimer, shown in a schematic representation, are colored in deep teal (subunit A) and violet (subunit B). The kynurenine substrate (Kyn), and the protein residues within 5 Å distance of the kynurenine substrate are shown. The 2Fo-Fc electron density map covering the kynurenine and cofactor (PMP) is shown contoured at the 0.7 and 1 sigma, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The hKAT-II architecture, however, represents a very different subfold type from the P. horikoshii KAT-II (pKAT-II) although they share 40% structural similarity (Z-score) with each other (39Chon H. Matsumura H. Koga Y. Takano K. Kanaya S. Proteins. 2005; 61: 685-688Crossref PubMed Scopus (17) Google Scholar, 60Holm L. Sander C. Science. 1996; 273: 595-603Crossref PubMed Scopus (1289) Google Scholar). Upon superposing the native hKAT-II structure onto the pKAT-II structure (Fig. 2c), we identified the major difference in protein folding to be at their N-terminal residues. In the hKAT-II structure, there are two β-strands (b1 and b2) formed between Lys-49 and Glu-65 with a turn in residues 55 to 59. The region shows very good electron density (Fig. 2b), which allowed us to unambiguously assign the observed conformations. The two-stranded β-sheet structure is critical in forming a different conformation for hKAT-II (Fig. 2), because pKAT-II forms an α-helix (a2) in this region (Fig. 2c). In addition, the residues 1-49 of hKAT-II reside in the position that is occupied by the N-terminal residues from the other subunit of the biological dimer in other fold-type I aminotransferases. Here, the two-stranded β-sheet structure in hKAT-II is unique with respect to the other type I aminotransferases. Because no similar conformation has been found in all subclasses of the fold-type I PLP-dependent enzymes, we propose that hKAT-II represents a novel subclass in this group based upon its unique fold of the first 64 N-terminal residues. In the crystal, a homodimer of hKAT-II is found with the subunits related by a 2-fold non-crystallographic axis. Both subunits are involved in building up the two identical active sites of the hKAT-II dimer, which are positioned ∼20-Å apart (the distance between two cofactors). The pattern of subunit-subunit interactions is extensive, and residues from the N-terminal part (residues 9-49) from one subunit and a small and large domain from another subunit. The two strands from both subunits (β-strand 1, b1 in Fig. 2) form an inter-subunit β-sheet. This two-strand interaction is unique in hKAT-II structure. Active Site of hKAT-IIResidual electron density clearly revealed the presence of covalently bound PLP in the cleft situated at the interface of the subunits in the biological dimer (Fig. 4). The C4A atom of PLP is covalently attached to the NZ atom of Lys-263 through the formation of an internal Schiff base, and the internal aldimine gives rise to residue LLP263, represented as sticks in Fig. 4. The PLP pyridoxal ring is stacked between residues Tyr-142 and Pro-232 by hydrophobic interactions, and the C2A atom of PLP exhibits a hydrophobic interaction with the Val-197 side chain. The side chains of Tyr-233 and Asp-230 are hydrogen-bonded to O3 and N1 of the pyridoxal, respectively. The phosphate moiety of PLP is anchored by polar interactions with the peptide amide groups of residues Gly-116 and Ser-117 as well as by the side chains of Ser-117, Gln-118, Ser-262, Arg-270, and another subunit residue Tyr-74*. All residues involved in PLP binding in hKAT-II are identical to those involved in the PLP binding sites in pKAT-II. However, it is worthwhile to mention that the stacking interactions of Pro-232 with the re face of the pyridine ring of PLP in KAT-II enzymes has not been observed in other available aminotransferase structures. Substrate Recognition and CatalysisInspection of the crystal structure of the hKAT-II:kynurenine complex revealed that the substrate lies near the N1 atom of PMP, but kynurenine and the cofactor do not form an external aldimine. Several residues, including Arg-399, Tyr-142, Ser-143, and Asn-202 from one subunit, and Gly-39*, Leu-40*, Ile-19*, Arg-20*, and Tyr-74* from opposite subunit, define the substrate-binding site and contact the kynurenine molecule. The carboxylic group of the kynurenine substrate forms a salt bridge with the guanidinium group of Arg-399. The salt bridge is fixed by hydrogen-bonding interactions with the NH of Asn-202 from one subunit and the side chain of Gly-38* from another subunit at both sides of the salt bridge. The ring of Tyr-74* has a weak hydrophobic interaction with the phenyl ring of kynurenine, and its hydroxy group forms a weak hydrogen bond with the O2 atom of kynurenine. Gln-38* and Gly-39* are located at the turning point of the loop that dives into and partially plugs the enzyme active site, thus shielding the substrate-binding pocket from bulk solvents (Fig. 5). Upon superposing the native hKAT-II structure onto the hKAT-II:kynurenine complex structure (Fig. 6), we identified a major chain conformational change involving residues 16 to 31. By binding the substrate kynurenine, this N-terminal part moves from one side of the active center to the other, leaves space for the substrate aminophenyl side to bind (Fig. 6). It also encapsulates the carboxylic side of the substrate within the center; thereby, the movement of residues 16-31 is not only for substrate binding but also for shielding the substrate-binding pocket from bulk solvents. This conformational change also involves some side-chain movement, including Tyr-74*, Tyr-142, and Ser-143, which shift toward the substrate (Fig. 6). At the same time, the pyridine ring of PMP also tilts toward the substrate and leaves the C4 atom vulnerable for substrate attack. It is well known that by binding substrates, aspartate aminotransferase changes its conformation from an open to a closed form (61Rhee S. Silva M.M. Hyde C.C. Rogers P.H. Metzler C.M. Metzler D.E. Arnone A. J. Biol. Chem. 1997; 272: 17293-17302Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 62Miyahara I. Hirotsu K. Hayashi H. Kagamiyama H. J. Biochem. (Tokyo). 1994; 116: 1001-1012Crossref PubMed Scopus (43) Google Scholar, 63Malashkevich V.N. Strokopytov B.V. Borisov V.V. Dauter Z. Wilson K.S. Torchinsky Y.M. J. Mol. Biol. 1995; 247: 111-124Crossref PubMed Scopus (83) Google Scholar, 64McPhalen C.A. Vincent M.G. Jansonius J.N. J. Mol. Biol. 1992; 225: 495-517Crossref PubMed Scopus (188) Google Scholar, 65Okamoto A. Higuchi T. Hirotsu K. Kuramitsu S. Kagamiyama H. J. Biochem. (Tokyo). 1994; 116: 95-107Crossref PubMed Scopus (144) Google Scholar, 66Jager J. Moser M. Sauder U. Jansonius J.N. J. Mol. Biol. 1994; 239: 285-305Crossref PubMed Scopus (175) Google Scholar), which involves a large-scale conformational change (domain-domain rotation). In hKAT-II, however, there is no large scale conformational change upon binding of the substrate. In summary, KAT-II is a principal aminotransferase involved in KYNA production in mammalian and human brains; therefore, it is apparently a potential regulatory target for maintaining physiological concentrations of brain KYNA. We report herein the crystal structures of hKAT-II and its complex with kynurenine. The presence of two-stranded β-sheet structures formed from N-terminal residues in the hKAT-II crystal structures separates it from other type I fold PLP-dependent enzymes. In addition, two-stranded β-sheet interaction of two subunits partially contributes to the formation of a homodimer. Moreover, the major main chain shift of residues 16-31 is essential for substrate binding and catalysis, which distinguishes it from most other typical type I fold aminotransferases whose substrate binding and catalysis largely depend upon domain-domain rotation. The structures of native hKAT-II and its complex with kynurenine provide an important molecular basis toward a comprehensive understanding of the substrate binding and catalysis in hKAT II, thus making it possible to work with structure ligand-based drug design targeting of this important enzyme in humans.