2
792 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Han et al.
concentration of 10 mg/mL in 10 mM potassium phosphate buffer
(7) Alkondon, M.; Pereira, E. F.; Yu, P.; Arruda, E. Z.; Almeida, L. E.;
Guidetti, P.; Fawcett, W. P.; Sapko, M. T.; Randall, W. R.; Schwarcz,
R.; Tagle, D. A.; Albuquerque, E. X. Targeted deletion of the
kynurenine aminotransferase ii gene reveals a critical role of endog-
enous kynurenic acid in the regulation of synaptic transmission via
alpha7 nicotinic receptors in the hippocampus. J. Neurosci. 2004, 24
(19), 4635–4648.
(
pH 6.8). The specific activity assay for KAT was also performed
35
according to a previous method.
hKAT I Crystallization. The crystals were grown through
hanging drop vapor diffusion methods with the volume of the
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
(8) Stone, T. W. Kynurenic acid blocks nicotinic synaptic transmission
to hippocampal interneurons in young rats. Eur. J. Neurosci. 2007,
crystallization buffer contained 22% PEG 4000, 0.2 M sodium
acetate, 0.1 M Tris, pH 8.5. hKAT I/IAC complex was cocrystal-
lized by adding 2.5 mM IAC and 4% glycerol (GOL) to the
crystallization buffer, and hKAT I/GOL complex was cocrystallized
by adding 5% GOL to the above crystallization buffer.
2
5 (9), 2656–2665.
(
9) Wang, J.; Simonavicius, N.; Wu, X.; Swaminath, G.; Reagan, J.; Tian,
H.; Ling, L. Kynurenic acid as a ligand for orphan G protein-coupled
receptor GPR35. J. Biol. Chem. 2006, 281 (31), 22021–22028.
(10) Beal, M. F.; Matson, W. R.; Swartz, K. J.; Gamache, P. H.; Bird,
E. D. Kynurenine pathway measurements in Huntington’s disease
striatum: evidence for reduced formation of kynurenic acid. J. Neu-
rochem. 1990, 55 (4), 1327–1339.
11) Guidetti, P.; Reddy, P. H.; Tagle, D. A.; Schwarcz, R. Early
kynurenergic impairment in Huntington’s disease and in a transgenic
animal model. Neurosci. Lett. 2000, 283 (3), 233–235.
(12) Widner, B.; Leblhuber, F.; Walli, J.; Tilz, G. P.; Demel, U.; Fuchs,
D. Tryptophan degradation and immune activation in Alzheimer’s
disease. J. Neural Transm. 2000, 107 (3), 343–353.
13) Schwarcz, R.; Rassoulpour, A.; Wu, H. Q.; Medoff, D.; Tamminga,
C. A.; Roberts, R. C. Increased cortical kynurenate content in
schizophrenia. Biol. Psychiatry 2001, 50 (7), 521–530.
14) Erhardt, S.; Blennow, K.; Nordin, C.; Skogh, E.; Lindstrom, L. H.;
Engberg, G. Kynurenic acid levels are elevated in the cerebrospinal
fluid of patients with schizophrenia. Neurosci. Lett. 2001, 313 (1-2),
96–98.
Data Collection and Processing. Individual hKAT I crystals
were cryogenized in crystallization buffer containing 20% glycerol
or 25% PEG 300 as a cryoprotectant. Diffraction data of hKAT I
crystals were collected at the Brookhaven National Synchrotron
Light Source beamline X29A (λ ) 1.0809 Å). Data collection was
done using an ADSC Q315 CCD detector. All data were indexed
(
5
0
and integrated using HKL software. Scaling and merging of
51
diffraction data were performed using the program SCALEPACK.
(
The parameters of the crystals and information regarding data
collection are described in Table 1.
Structure Determination. The structures of the hKAT I
complexes were determined by the molecular replacement method
using the published hKAT I structure without any ligands or water
(
2
4
molecules (Protein Data Bank code 1W7L). The program
5
2
(15) Erhardt, S.; Schwieler, L.; Nilsson, L.; Linderholm, K.; Engberg, G.
Molrep in the CCP4 suite was employed to calculate both cross-
rotation and translation function of the model. The initial model
was subjected to iterative cycles of crystallographic refinement with
The kynurenic acid hypothesis of schizophrenia. Physiol. BehaV. 2007,
9
2 (1-2), 203–209.
(
(
(
16) Guillemin, G. J.; Kerr, S. J.; Brew, B. J. Involvement of quinolinic
acid in AIDS dementia complex. Neurotoxic. Res 2005, 7 (1-2), 103–
123.
5
3
Refmac 5.2 and graphic sessions for model building using the
5
4
program O. The substrate molecules were modeled when the R
17) Colombari, E.; Sato, M. A.; Cravo, S. L.; Bergamaschi, C. T.; Campos,
R. R., Jr.; Lopes, O. U. Role of the medulla oblongata in hypertension.
Hypertension 2001, 38 (3, Part 2), 549–554.
18) Ito, S.; Komatsu, K.; Tsukamoto, K.; Sved, A. F. Excitatory amino
acids in the rostral ventrolateral medulla support blood pressure in
spontaneously hypertensive rats. Hypertension 2000, 35 (1, Part 2),
413–417.
factor dropped to a value of 0.24 at full resolution for all hKAT I
structures based on both the 2F
o
- F
c
and F
o
c
- F electron density
maps. Solvent molecules were automatically added and refined by
5
5
using ARP/wARP together with Refmac 5.2.
Structure Analysis. Superposition of structures was done using
5
6
Lsqkab from the CCP4 suite. Figures were generated using
57
(
19) Kwok, J. B.; Kapoor, R.; Gotoda, T.; Iwamoto, Y.; Iizuka, Y.; Yamada,
N.; Isaacs, K. E.; Kushwaha, V. V.; Church, W. B.; Schofield, P. R.;
Kapoor, V. A missense mutation in kynurenine aminotransferase-1 in
spontaneously hypertensive rats. J. Biol. Chem. 2002, 277 (39), 35779–
Pymol. Protein and substrate interactions were also analyzed using
5
7
Pymol.
Acknowledgment. This work was carried out in part at the
National Synchrotron Light Source, Brookhaven National
Laboratory, and supported in part by the Intramural Research
Program of the institutes of NIDCR and NINDS at NIH. We
are grateful to Elizabeth Watson and Graham Richardson (Dr.
Jianyong Li’s laboratory, Department of Biochemistry, Virginia
Tech) for critical reading of this paper.
2
5782.
(20) McGoldrick, T. A.; Lock, E. A.; Rodilla, V.; Hawksworth, G. M. Renal
cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes
in primary cultures of human and rat proximal tubular cells. Arch.
Toxicol. 2003, 77 (7), 365–370.
21) Dekant, W.; Vamvakas, S.; Anders, M. W. Formation and fate of
nephrotoxic and cytotoxic glutathione S-conjugates: cysteine conjugate
beta-lyase pathway. AdV. Pharmacol. 1994, 27, 115–162.
22) Spencer, J. P.; Whiteman, M.; Jenner, P.; Halliwell, B. 5-s-Cysteinyl-
conjugates of catecholamines induce cell damage, extensive DNA base
modification and increases in caspase-3 activity in neurons. J. Neu-
rochem. 2002, 81 (1), 122–129.
(
(
Supporting Information Available: Figures S1-S4 showing
omit maps. This material is available free of charge via the Internet
at http://pubs.acs.org.
(
23) Cooper, A. J.; Pinto, J. T.; Krasnikov, B. F.; Niatsetskaya, Z. V.; Han,
Q.; Li, J.; Vauzour, D.; Spencer, J. P. Substrate specificity of human
glutamine transaminase K as an aminotransferase and as a cysteine
S-conjugate beta-lyase. Arch. Biochem. Biophys. 2008, 474 (1), 72–
References
(
1) Leeson, P. D.; Iversen, L. L. The glycine site on the NMDA receptor:
structure-activity relationships and therapeutic potential. J. Med.
Chem. 1994, 37 (24), 4053–4067.
8
1.
(24) Rossi, F.; Han, Q.; Li, J.; Li, J.; Rizzi, M. Crystal structure of human
kynurenine aminotransferase I. J. Biol. Chem. 2004, 279 (48), 50214–
50220.
(25) Jansonius, J. N. Structure, evolution and action of vitamin B6-
dependent enzymes. Curr. Opin. Struct. Biol. 1998, 8 (6), 759–769.
(26) Eliot, A. C.; Kirsch, J. F. Pyridoxal phosphate enzymes: mechanistic,
structural, and evolutionary considerations. Annu. ReV. Biochem. 2004,
73, 383–415.
(27) Han, Q.; Gao, Y. G.; Robinson, H.; Ding, H.; Wilson, S.; Li, J. Crystal
structures of Aedes aegypti kynurenine aminotransferase. FEBS J.
2005, 272 (9), 2198–2206.
(28) Goto, M.; Omi, R.; Miyahara, I.; Hosono, A.; Mizuguchi, H.; Hayashi,
H.; Kagamiyama, H.; Hirotsu, K. Crystal structures of glutamine:
phenylpyruvate aminotransferase from Thermus thermophilus HB8:
induced fit and substrate recognition. J. Biol. Chem. 2004, 279 (16),
16518–16525.
(
2) Perkins, M. N.; Stone, T. W. An iontophoretic investigation of the
actions of convulsant kynurenines and their interaction with the
endogenous excitant quinolinic acid. Brain Res. 1982, 247 (1), 184–
1
87.
(
(
(
(
3) Stone, T. W.; Perkins, M. N. Actions of excitatory amino acids and
kynurenic acid in the primate hippocampus: a preliminary study.
Neurosci. Lett. 1984, 52 (3), 335–340.
4) Birch, P. J.; Grossman, C. J.; Hayes, A. G. Kynurenic acid antagonises
responses to NMDA via an action at the strychnine-insensitive glycine
receptor. Eur. J. Pharmacol. 1988, 154 (1), 85–87.
5) Pereira, E. F.; Hilmas, C.; Santos, M. D.; Alkondon, M.; Maelicke,
A.; Albuquerque, E. X. Unconventional ligands and modulators of
nicotinic receptors. J. Neurobiol. 2002, 53 (4), 479–500.
6) Hilmas, C.; Pereira, E. F.; Alkondon, M.; Rassoulpour, A.; Schwarcz,
R.; Albuquerque, E. X. The brain metabolite kynurenic acid inhibits
alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic
receptor expression: physiopathological implications. J. Neurosci.
(29) Wogulis, M.; Chew, E. R.; Donohoue, P. D.; Wilson, D. K.
Identification of formyl kynurenine formamidase and kynurenine
aminotransferase from Saccharomyces cereVisiae using crystallo-
2
001, 21 (19), 7463–7473.