226
H.-W. KIM et al.
Table 1. Substrate Specificity of PhGAD from P. horikoshii
its activity was very stable against heat inactivation.
It survived for prolonged periods at 85 ꢀC. Its half-life
at 95 ꢀC was 3 h.
Substrates
Km (mM)
Specific activity (kcat) (1/s)
L-glutamate
L-aspartate
Cysteate
3:9 ꢃ 0:4
1:2 ꢃ 0:1
2:2 ꢃ 0:3
32:6 ꢃ 5:4
ND
0:26 ꢃ 0:03
0:34 ꢃ 0:02
0:65 ꢃ 0:07
0:03 ꢃ 0:00
ND
GAD enzymes have been reported from widely varied
organisms, from bacteria to mammals. Bacterial and
plant GADs share similar features in their biochemical
properties and their functions, such as specific activity
against glutamate, acidic optimal pH, and their response
mechanisms to environmental stress.8,18) In contrast to
these enzymes, PhGAD showed biochemically broad
substrate specificity and optimal activity under weak
alkali pH conditions. Although its amino acid sequence
did not share homology with that of the mammalian
enzyme, these biochemical properties closely resembled
those of the mammalian enzyme, which has a neutral
optimal pH, and also showed activity against various
substrates, such as cysteate, cysteine sulfite, and L-
aspartate-like PhGAD.19,20) As a representative func-
tion of bacterial GAD, E. coli GAD is known to be
involved in resistance to acidic environments, but
PhGAD might be not relevant to this function in view
of its alkaline optimal pH. Consequently, the biochem-
ical properties of PhGAD suggest that the enzyme
participates in the metabolic pathways of various amino
acids according to the internal milieu of the host cell.
This is the first report on archaeal hyperthermophilic
GAD. This enzyme has lower GAD activity than other
GADs, but its hyperthermostability is fascinating in light
of its potential for the industrial production of GABA.
However, we continue our studies to improve the
activity of PhGAD using protein engineering techniques.
Cysteine sulfite
L-tyrosine
D-glutamate
D-aspartate
ND
ND
ND
ND
ND, No activity was detected.
firmed that enzymatic recovery from the aggregate was
affected by ionic-strength-dependent solubility, and the
solubilized GAD exhibited activity.
Purification of PhGAD was performed using 20 mM
Tris buffer (pH 7.5) containing 0.4 M NaCl and 1 mM
PLP following heat treatment (for 30 min at 85 ꢀC).
Ammonium sulfate was dissolved into the supernatant
after heat treatment to 1.7 M, and applied to a HiTrap
phenyl HP(Amersham, Piscataway, NJ) chromatography
column equilibrated with 20 mM Tris–HCl (pH 7.5)
containing 0.25 mM PLP, 0.4 M NaCl, and 1.7 M ammo-
nium sulfate. The enzyme was eluted using a linear
decrease in the ammonium sulfate concentration in the
buffer from 1.7 to 0 M. The enzyme solution was
collected and dialyzed against 20 mM Tris–HCl (pH 7.5)
containing 0.25 mM PLP and 0.4 M NaCl. The purified
enzyme ran as a single protein band as analyzed by
SDS–PAGE, indicating that it had been purified to
homogeneity.
The molecular weight of the enzyme, which was
about 43 kDa based on DNA sequence, was estimated to
be 45 kDa and 42 kDa by gel filtration and SDS–PAGE,
respectively. The structure of PhGAD appears to
be monomer. In contrast, most GADs reported in
literature exist in a multimeric form, such as dimer or
hexamer.8,15) Only GAD from the fungus Neurospora
crassa has been reported to be expressed in monomer
form.16)
GAD activity was measured using a modified version
of the method described by Okada et al.17) The reaction
mixture (0.1 ml) consisted of 50 mM glutamate, 0.25 mM
PLP, 0.4 M NaCl, and 0.1 M sodium phosphate (pH 8.0).
The enzyme reaction was performed by incubating the
mixture for 60 min at 85 ꢀC. Then 20 ml aliquots of the
mixture were dried and treated with phenyl-isothiocya-
nate. The resulting phenylthiocarbamoyl-gamma-amino-
butyric acid was measured using a high-performance
liquid chromatography system (HPLC; Tosoh, Tokyo)
with a column (4:6 ꢁ 150 mm, Wakosil-PTC; Wako
Pure Chemical Industries, Kyoto). For the substrate
specificities of PhGAD, the decarboxylation activities
toward L-aspartate, cysteinate, L-tyrosine, and cysteine
sulfinate were determined by measuring the concentra-
tions of the decarboxylation products of their respective
reactions under the same reaction conditions: ꢂ-alanine,
taurine, tyramine, and hypotaurine. Compared with the
decarboxylation activities toward the various substrates,
the specific activities toward cysteate and L-aspartate
represented slightly higher values than L-glutamate; no
activity against L-tyrosine was found (Table 1).
Acknowledgments
This work was supported by a Korea Research
Foundation Grant, funded by the Korean Government
(MOEHRD) (KRF-2005-214-D00276).
References
1) Roberts, E., and Frankel, S., gamma-Aminobutyric acid in
brain: its formation from glutamic acid. J. Biol. Chem., 187, 55–
63 (1950).
2) Manyam, B. V., Katz, L., Hare, T. A., Kaniefski, K., and
Tremblay, R. D., Isoniazid-induced elevation of CSF GABA
levels and effects on chorea in Huntington’s disease. Ann.
Neurol., 10, 35–37 (1981).
3) Jakobs, C., Jaeken, J., and Gibson, K. M., Inherited disorders of
GABA metabolism. J. Inherit. Metab. Dis., 16, 704–715 (1993).
4) Hagiwara, H., Seki, T., and Ariga, T., The effect of pre-
germinated brown rice intake on blood glucose and PAI-1 levels
in streptozotocin-induced diabetic rats. Biosci. Biotechnol.
Biochem., 68, 444–447 (2004).
5) Siragusa, S., De Angelis, M., Di Cagno, R., Rizzello, C. G.,
Coda, R., and Gobbetti, M., Synthesis of gamma-aminobutyric
acid by lactic acid bacteria isolated from a variety of Italian
cheeses. Appl. Environ. Microbiol., 73, 7283–7290 (2007).
6) Kato, Y., Furukawa, K., and Hara, S., Cloning and nucleotide
sequence of the glutamate decarboxylase-encoding gene gadA
from Aspergillus oryzae. Biosci. Biotechnol. Biochem., 66,
2600–2605 (2002).
7) Buss, K., Drewke, C., Lohmann, S., Piwonska, A., and Leistner,
E., Properties and interaction of heterologously expressed
glutamate decarboxylase isoenzymes GAD(65 kDa) and
GAD(67 kDa) from human brain with ginkgotoxin and its
50-phosphate. J. Med. Chem., 44, 3166–3174 (2001).
8) Capitani, G., De Biase, D., Aurizi, C., Gut, H., Bossa, F., and
Grutter, M. G., Crystal structure and functional analysis of
Escherichia coli glutamate decarboxylase. EMBO J., 22, 4027–
4037 (2003).
The range of pH at which PhGAD was active and
stable was determined with glutamate as a substrate.
GAD activity was observed at maximum at pH 8.0, and
was stable at weak alkalinity, of 8.0–8.5. The optimum
temperature of the enzyme was higher than 97 ꢀC, and