References
dithiothreitol and 50 µM pyridoxal 5'-phosphate). The protein
solution was concentrated to 10 mg/mL for crystallization
experiments.
1. Foltyn V. N., Bendikov I., Miranda J. D., Panizzutti R.,
Dumin E., Shleper M., Li P., Toney M. D., Kartvelishvily E.,
Wolosker H. Serine racemase modulates intracellular D-
serine levels through an α,β-elimination activity. J. Biol.
Chem. 2005; 280; 1754-1763.
Optimal Crystals of SR were obtained at 20 °C by the sitting
drop vapor diffusion method. Diffraction-quality crystals were
obtained with a reservoir solution of 10% PEG8000, 5 mM
MgCl2, 10% ethylene glycol and 0.1 M Bis-Tris (pH 6.0).
Crystals were cryoprotected with 30% ethylene glycol and frozen
by dunking in liquid nitrogen. The diffraction data were collected
at 100K on Beamline BL26B1 at SPring8 (Harima, Japan) with a
1.0 Å wavelength. The dataset was indexed and integrated with
the software MOSFLM17 and scaled with the Scala included in
the CCP4 crystallographic package18. The crystallographic phase
was determined using the molecular replacement method
implemented in Phaser19, using a mutant of SR as a search model
(PDB 3L6B). The structure was manually adjusted with the
graphic software Coot20 and refined with Phoenix21. The final
models of SR had 97.2% of residues in the core areas of the
Ramachandran plot with no residues in disallowed regions. The
data collection and refinement statistics are presented in Table S1.
Structural coordinates have been deposited in the Protein Data
Bank (PDB Accession number 5X2L).
2. Hashimoro A., Nishikawa T., Hayashi T., Fujii N., Harada
K., Oka T., Takahashi K. The presence of free D-serine in rat
brain. FEBS lett. 1992; 296: 33-36.
3. Inoue R., Hashimoto K., Harai T., Mori H. NMDA- and β-
amyloid1-42-induced neurotoxicity is attenuated in serine
racemase knock-out mice. J. Neurosci. 2008; 28: 14486-
14491.
4. Basu A. C., Tsai G. E., Ma C. L., Ehmsen J. T., Mustafa A.
K., Han L., Jiang Z. I., Benneyworth M. A., Froimowitz M.
P., Lange N., Snyder S. J., Bergeron R., Coyle J. T. Targeted
disruption of serine racemase affects glutamatergic
neurotransmission and behanior. Mol. Psychiatry. 2009; 14:
719-727.
5. Labrie V., Fukumara R., Rastogi A., Fick L. J., Wang W.,
Boutros P. C., Kennedy J. L., Semeralul M. O., Lee. F. H.,
Baker G. B., Belsham D. D., Barger S. W., Gondo Y., Wong
A. H. C., Roder J. C. Serine racemase is associated with
schizophrenia susceptibility in humans and in a mouse
model. Hum. Mol. Genet. 2009; 18: 3227-3243.
For the assay of compounds 1, 2, 5A, B, 9A-H, 12A, B, 15A,
B, 19 and 21, enzyme activities were examined as described
previously14 using 1.25 µg wild-type SR in a final volume of 125
µL of 100 mM HEPES (pH 8.0), 10 µM PLP, 1 mM MgCl2, 5
mM DTT, 1 mM ATP, 20 mM L-Ser, and 1 mM inhibitors. The
reaction mixtures were incubated for 30 min at 37°C. The
reaction mixtures (25 µL) were further incubated in 100 mM
HEPES (pH 8.0), 10 µM PLP, and recombinant Dsd1 in a final
volume of 50 µL for 30 min at 30°C. The reaction mixtures were
mixed with 50 µL of 0.05% DNP in 2 M HCl aq. and incubated
for 5 min at 30 °C. To the reaction mixtures, 100 µL of EtOH and
125 µL of 10 M NaOH aq. were sequentially added and then the
mixtures were incubated for 10 min at room temperature.
Absorbance at 515 nm of the resultant hydrazine was measured.
In our assay condition, the Km value for SR to produce D-serine
from L-serine was 6.6 mM. (Supplementary material) We also
examined the D-serine production by SR with 30 min incubation
and found the velocity of the reaction is first order. Our assay
condition is based on the previous report measuring the activity
of recombinant mouse SR10. The activity of the inhibitors was
evaluated with the percentage of the D-serine production
compared with that without inhibitors.
6. Bliss T. V. P., Collingridge G. L. A synaptic model of
memory: long-term potentiation in the hippocampus. Nature
1993; 361: 31-39.
7. Komuro H., Pakic P. Modulation of neuronal migration by
NMDA receptors. Science 1993; 260: 95-97.
8. Lancelot E., Beal M. F. Glutamate toxicity in chronic
neurodegenerative disease. Prog. Brain. Res. 1998; 116:
331-347.
9. Mustafa A. K., Ahmad A. S., Zeynalov E., Gazi S. K., Sikka
G., Ehmsen J. T., Barrow R. K., Coyle J. T., Snyder S. H.,
Dore S. Serine racemase deletion protects against cerebral
ischemia and excitotoxicity. J. Neurosci. 2010; 30: 1413-
1416.
10. Strisovsky K., Jiraskova J., Mikulova A., Rulisek L.,
Konvalinka J. Dual substrate and reaction specificity in
mouse serine racemase: Identification of high-affinity
dicarboxylate substrate and inhibitors and analysis of the β-
eliminate activity. Biochemistry. 2005; 44: 13091-13100.
We synthesized 15 derivatives of 1, and evaluated their
inhibitory activities against wild-type SR. Among the synthesized
compounds, the derivative 9C was a potent inhibitor of wild-type
SR. In future, a novel inhibitor of SR should be designed based
on the structure of wild-type SR.
11. Kim P. M., Aizawa H., Kim P. S., Huang A. S.,
Wickramasinghe S. R., Kashani A. H., Barrow R. K.,
Huganir R. L., Ghosh A., Snyder S. H. Serine racemase:
Activation by glutamate neurotransmission via glutamate
receptor interacting protein and mediation of neuronal
migration. Proc. Natl. Acad. Sci. USA. 2005; 102: 6032-
6041.
A. Supplementary data
12. Dixon S. M., Li P., Liu R., Wolosker H., Lam K. S., Kurth
M. J., Toney M. D. Slow-binding human serine racemase
inhibitors from high-throughput screening of combinatorial
libraries. J. Med. Chem. 2006; 49: 2388-2397.
Supplementary data (In silico screening, Modeling of binding
mode, Crystal structure, Characterization of recombinant wild-
type SR, Experimental informations, Copies of 1H- and 13C-NMR
spectra) were added as a Supporting Information.
13. Vorlova B., Nachtigallova D., Jiraskova-Vanickova J., Ajani
H., Jansa P., Rezac J., Fanfrlik J., Otyepka M., Hobza P.,
Konvalinka J., Lepsik M. Malonate-based inhibitors of
mammalian serine racemase: Kinetic characterization and