Download Arg305 of Streptomyces l-glutamate oxidase plays a crucial role for

Biochemical and Biophysical Research Communications 417 (2012) 951–955
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Arg305 of Streptomyces L-glutamate oxidase plays a crucial role
for substrate recognition
Tomohiro Utsumi a,1, Jiro Arima b,1, Chika Sakaguchi a, Takashi Tamura a, Chiduko Sasaki c,
Hitoshi Kusakabe d, Shigetoshi Sugio c, Kenji Inagaki a,⇑
a
Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
Innovation Center Yokohama Center, Mitsubishi Chemical Corporation, Aoba-ku, Yokohama 227-8502, Japan
d
Enzyme Sensor Co., Ltd., Liaison Center 311, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan
b
c
a r t i c l e
i n f o
Article history:
Received 29 November 2011
Available online 16 December 2011
Keywords:
L-Glutamate oxidase
Streptomyces
Substrate recognition
Substrate specificity
a b s t r a c t
Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp.
X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking
simulation using L-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and
Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX
toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However,
the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various
L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants
of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The
results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the
interaction of enzyme and side chain of substrate.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
L-Glutamate oxidase (LGOX; EC 1.4.3.11) catalyzes the oxidative
deamination of an L-glutamate to a 2-ketoglutarate via an imino
acid intermediate. LGOXs of several kinds have been identified
from the genus Streptomyces [1–4]. Among them, an enzyme from
Streptomyces sp. X-119-6 is the sole commercially available enzyme that is useful for biosensors: it has strict substrate specificity
and high stability. LGOX is a useful analytical tool for the quantitative assaying of L-glutamate existing in food, in a fermentation
process, and in the brain acting as the principal excitatory neurotransmitter. In addition, microfluidic biosensors using LGOX are
applicable for the detection of glutamic oxaloacetic transaminase,
glutamic pyruvic transaminase and c-glutamyl transpeptidase
activities, which are diagnostic markers of liver function [5,6].
As a similar enzyme, L-amino acid oxidases (LAOs) have been
isolated from various sources [7–13]. General LAOs (for example
LAO from snake venome) catalyze the oxidation of a variety of
Abbreviations: LGOX, L-glutamate oxidase; LAO, L-amino acid oxidase; KPB,
potassium phosphate buffer; MBTH, 3-methyl-2-benzothiazolone hydrazone
hydrochloride.
⇑ Corresponding author. Fax: +81 86 251 8299.
E-mail address: [email protected] (K. Inagaki).
1
These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.12.033
L-amino
acids. Meanwhile, several LAOs with strict substrate
specificity have been identified: L-lysine-a-oxidase [14], L-phenylalanine oxidase [15,16], and L-aspartate oxidase [17]. They have an
identical catalytic mechanism but different affinities for substrate.
Elucidation of the mechanism for substrate recognition create the
potential of a molecular design of this class of the enzyme; that
is, a development of potent biocatalyst for L-amino acid sensor
through modification of their properties using protein engineering
techniques. Particularly, sensor for measurement of L-histidine and
L-arginine has high demand in the food and medical fields; that is,
L-histidine is one of the essential amino acid for human that has a
beneficial effect on anti-obesity [18] and L-arginine is one of the
semiessential amino acid for human that is important for removal
of ammonia from human body.
Recently, we solved the crystal structure of LGOX (PDB code:
2E1M) [19]. The enzyme has deeply buried active site with unique
structure. The arrangement of the residues composing the active
sites of LGOX and LAO are different from each other. Although the
above difference is considered to be associated with their difference
in substrate specificity, the details of the mechanism for substrate
recognition remains unknown. For this study, we selected Arg305
as candidates of the residues associating with the substrate recognition by docking study. Substrate specificity of Arg305 variants
changed greatly. Furthermore, based on a comparative kinetic
952
T. Utsumi et al. / Biochemical and Biophysical Research Communications 417 (2012) 951–955
study, the mechanism for substrate recognition of LGOX is
discussed.
with the equilibrium buffer. Then the enzyme was eluted using the
same buffer containing 10 mM maltose.
2. Materials and methods
2.6. Maturation of LGOX and its variants by protease digestion
2.1. Docking study
The purified LGOX and its variants by Amylose resin were
single-chain LGOX precursor fused with maltose binding protein.
The digestion by metallo-protease from Streptomyces was necessary for maturation of LGOX [22] (the mature form of LGOX has
a2b2c2 structure). To maturation of them, the purified enzymes
were treated with the metallo protease from Streptomyces griseus
(Sigma–Aldrich Corp., St. Louis, MO, USA) at 30 °C for 24 h. Then
it was heated at 50 °C for 30 min to inactivate the protease. After
centrifugation, the solution was loaded onto a column (DEAEToyopearl 650; Tosoh Corp., Tokyo, Japan) equilibrated with
20 mM KPB (pH 7.4) containing 0.1 M NaCl. After the column
was washed with the same buffer, the bound protein was eluted
with 20 mM KPB (pH 7.4) containing 0.3 M NaCl. The eluate was
pooled and dialyzed against 20 mM KPB (pH 7.4); the dialysate
was used as the mature enzyme.
The docking study was performed using Internal Coordinate
Mechanics (ICM) software (Molsoft L.L.C., La Jolla, USA), a program
for comparative protein structure modeling and interactive docking. The structure of L-glutamate was built into the software, and
simulation of interactive docking was performed using the rigid
form of LGOX and flexible form of L-glutamate. The simulation
was repeated 150 times with the calculation of total energy using
biased probability Monte-Carlo method as calculated before the
conformation was stored [20].
2.2. Enzyme assays
LGOX activity assay involved the determination of 2-ketoglutarate formed with 3-methyl-2-benzothiazolone hydrazone hydrochloride as described previously (MBTH method) [21]. One unit
of LGOX activity was taken as the amount of the enzyme that
liberated 1 lmol of 2-ketoglutarate per minute.
2.3. Construction of an expression vector
The full-sized gene encoding LGOX was amplified using PCR
from the chromosomal DNA of Streptomyces sp. X-119-6 using
the following primers: 50 -CCACACCGGGGCCGAATTCATGAACGAGAT-30 and 50 -AGGTACTCGGCCACCCTGCAGGTC-30 (the underlined
regions are EcoR I and Pst I). The PCR product was separated by
agarose gel electrophoresis and digested by EcoR I and Pst I, then
subcloned into the EcoR I and Pst I gap of pMal-c2 (yielding
pGox-mal1).
2.4. Site directed mutagenesis
Site-directed mutagenesis was conducted by inverse PCR using
a pair of oligonucleotide primers containing a point mutation (see
Table S1 in the supplemental material) and the pGOx-mal1. The
PCR program was as follows: 15 cycles of 10 s at 98 °C, 30 s at
65 °C, and 10 min at 68 °C. The PCR product was treated with DpnI
at 37 °C for 1 h. Then the PCR product was transfected into competent cell of Escherichia coli TOP10 according to the manufacturer’s
protocol. After the confirmation of the sequence, the extracted
plasmid was transformed into E. coli JM109.
2.5. Expression and purification of the recombinant LGOX and its
variants fused with maltose binding protein E. coli
JM109 cells harboring pGOx-mal1or the plasmid for mutant
LGOX expression were cultivated at 22 °C for 24 h in 3 l of 2 TY
medium. Expression of the LGOX was induced by incubation at
22 °C with 0.5 mM IPTG for 24 h. The harvested cells were suspended in 20 mM potassium phosphate buffer (KPB) (pH 7.4),
and then disrupted by ultrasonication on ice. After removal of
the cell debris, the supernatant was brought to 40% saturation with
ammonium sulfate. The resultant supernatant was brought to 60%
saturation with ammonium sulfate. The resultant precipitate was
dissolved in 20 mM Tris–HCl buffer (pH7.4) containing 200 mM
NaCl and 1 mM EDTA. Next, the solution was loaded onto an Amylose resin affinity column (New England Biolabs, Beverly, MA, USA)
equilibrated with the same buffer. The column was washed gently
2.7. Biochemical studies
The substrate specificity of wild-type and mutant LGOXs were
tested by the MBTH method with 0.5 mM L-tyrosine, 1 mM
L-tryptophan and L-aspartate, 5 mM L-leucine, and 10 mM other
substrates with the purified enzymes. Effects of pH on activity
were examined using Britton-Robinson buffer at a pH range of
4.0–12.0. The activities were measured by the MBTH method at
the corresponding pH.
3. Results and discussion
3.1. Conformational simulation of L-glutamate in active site of LGOX
The overall structure and local structure around the active site
of LGOX resembles that of LAO [19]. A previous report by Pawelek
et al. of the crystal structure of LAO interacted with o-aminobenzoate (AB) described that three AB molecules are visible within the
funnel of the LAO-AB complex [23]. Likewise, the structure of LGOX
interacted with ligand provide us with valuable information about
the mechanism of the substrate recognition of LGOX. However, the
LGOX structure interacting with a mimic of the natural substrate
was not analyzed because of the difficulty in obtaining crystals of
the LGOX–ligand complex. Therefore, we performed a docking
study using the LGOX structure as a receptor and L-glutamate as ligands to investigate the interaction of enzyme with a substrate.
By the simulation, we obtained numerous L-glutamate binding
forms around the active site. The L-glutamate molecules were
mainly present at three sites: the entrance to the active site, near
the active site, and the active center (see Fig. S1 in the Supplemental material). As shown in Fig. 1, the model at the active center
indicates that the hydrogen atom on Ca of L-glutamate points toward to isoalloxazine ring of FAD, and the distance between Ca
of L-glutamate and N5 of FAD is approximately 5 Å. Although this
is not sufficiently close for reaction, L-glutamate, the residues surrounding L-glutamate, and the FAD should be allowed to be flexible. Because it has suitable space and adequate basic residues in
the model of L-glutamate binding at the active center, LGOX might
have strict specificity for oxidation toward L-glutamate.
3.2. Mutation of Arg305, His312, and Trp564 of LGOX with Ala
In the binding model, the side chains of Arg305 and His 312 might
produce hydrogen bonding with the side chain of L-glutamate. In
T. Utsumi et al. / Biochemical and Biophysical Research Communications 417 (2012) 951–955
953
purification steps as described in Section 2. As judged by SDS–
PAGE, the wild-type LGOX and the mutants (R305A, R305L,
R305D, R305K LGOXs) could be produced and purified as described
in materials and methods (see Fig. S2 in the Supplemental
material).
By the investigation of specific activities of R305X mutants
toward glycine and 19 kinds of L-amino acids, we found that all
mutants catalyzed the oxidation of L-leucine, L-phenylalanine,
and L-histidine (Fig. 2). Moreover, the specificity of mutant enzymes toward respective L-amino acids was different from each
other. That is, R305A, R305L, R305K LGOXs showed the highest
activity toward L-histidine, however, the activity of R305D LGOX
toward L-arginine was higher than that toward L-histidine.
Fig. 1. Proposed model for L-glutamate binding at the active site of LGOX. The
residues associated with the L-glutamate binding are indicated as sticks with color
according to the atom type. L-Glutamate is shown as green. The isoalloxazine ring of
FAD is indicated by CPK with yellow; N5 is blue. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
this paper.)
addition, Trp564 also appears to be important for closing up the active site (Fig. 1). To investigate the roles of them in substrate specificity of LGOX, we constructed R305A, H312A, and W564A LGOXs by
site-directed mutagenesis. We used a pMal system to produce wildtype and mutant enzymes.
By using the cell free extract of them treated with metallo-protease from Streptomyces, the activities of mutants toward glycine and
19 kinds of L-amino acids were investigated. By the investigation, we
found that all mutants exhibited the activity for L-glutamate oxidation significantly lower than wild type; the mutation of Arg305 with
Ala resulted in about 20-fold decrease in L-glutamate oxidation, and
the activities of other mutants were even lower than that of R305A
LGOX (data not shown). The mutations of His312 and Trp564 with
Ala exerted little influence on substrate specificity. In contrast,
R305A LGOX catalyzed the oxidation of some L-amino acids, whereas
wild-type LGOX exhibits no oxidation activity toward L-amino acids
except L-glutamate.
3.3. Effect of mutation of Arg305 on substrate specificity of LGOX
Because the side chain of Arg behaves strong base, it seems to
interact strongly with the side chain of the substrate L-glutamate.
Therefore, we focused the residue Arg305 and investigated its roles
in substrate specificity of LGOX. We constructed R305X LGOX by
site-directed mutagenesis, then matured and purified it by three
3.4. Effect of pH on oxidative activity
To obtain information on the ionizable groups involved in the
formation of an ion bridge, the activities toward L-glutamate,
L-histidine, and L-arginine were determined at pH between 5 and
10. Fig. 3 shows the effect of pH on the initial velocities of the
parental and mutant enzymes using respective substrates. The
curves for L-histidine oxidation by mutant enzymes were narrower
than that for L-glutamate oxidation by wild type. In addition, the
pH range of L-histidine oxidation by R305K LGOX was shifted to
the alkaline side. The similar curve was observed in L-arginine oxidation by R305D LGOX. From these results, we speculate that the
formation of the ion bridge between the residue 305 of LGOX
and the substrate affects the pH dependence of oxidative activity.
That is, in such pH ranges, the side chain of the substrate tends
to form an ion bridge with the residue, and the formation of an
ion bridge leads to an increase in oxidative activity.
3.5. Kinetic study
We also characterized the kinetics of the wild-type and R305X
LGOXs; the results are summarized in Table 1. Although all mutation resulted in the loss of activity toward L-glutamate, the
catalytic efficiency toward L-histidine, L-phenylalanine, L-leucine,
or L-arginine of all mutants could be measured because of the
enhancement of activity toward them by the mutation. In terms
of catalytic efficiency toward L-histidine, R305L LGOX displayed
the highest value among the LGOX variants. The kcat value of
R305L LGOX for L-histidine oxidation showed only 1.3- or 2-fold
higher than those of other mutants. However, the Km value of
R305L for L-histidine oxidation was over 5.5-fold lower than those
of other mutant enzymes. The result indicates that the change of
Fig. 2. Oxidative activities of wild-type and R305X LGOXs toward L-leucine, L-phenylalanine, L-asparagine, L-histidine, L-arginine, and L-glutamate. The values are average of
two independent experiments.
954
T. Utsumi et al. / Biochemical and Biophysical Research Communications 417 (2012) 951–955
Table 1
Comparison of the kinetic parameters of wild-type and R305 variants for different
substrates.
LGOX variant
Substrate
Km (mM)
kcat (s1)
kcat/Km (M1 s1)
Wild-type
L-Glu
0.173
53.2
3.08 105
L-His
32.9
9.38
2.85 102
L-Phe
66.7
6.50
9.75 10
L-Leu
5.00
0.0795
1.59 10
L-His
5.70
12.1
2.12 103
L-Phe
31.3
10.9
3.48 102
L-Leu
5.92
0.901
1.52 102
L-Arg
7.20
1.65
2.29 102
L-His
38.5
5.27
1.37 102
L-Phe
90.9
0.954
1.05 10
L-His
32.8
4.84
1.48 102
L-Phe
13.9
0.619
4.45 10
L-Leu
35.7
0.254
7.11
R305A
R305L
R305D
R305K
the mutations. This shift leads to a change in the distance between
the substrate and the catalytic center (i.e. N5 atom of isoalloxazine
ring of FAD), resulting in a change in catalytic activity. Furthermore, in R305D LGOX, it is considered that an ion bridge was
formed between the side chain of the substrate L-arginine and that
of the substituted residue. In fact, only R305D LGOX had the catalytic activity toward L-arginine among the mutant enzymes (Fig. 2
and Table 1). It is thought that activity with L-arginine is enhanced
by a suitable distance because of the formation of an ion bridge,
that is, this formation of an ion bridge is a factor in the enhancement in catalytic activity.
In conclusion, we have found that Arg305 of LGOX plays a crucial role for substrate recognition. The substrate specificity of LGOX
was markedly changed by the substitution of Arg305 with other
amino acid residue. The results of kinetics and pH dependence
on activity indicate that Arg305 of LGOX is associated with the
interaction of enzyme and side chain of substrate. Through the
investigations, we obtained the enzymes that have oxidative activity toward L-histidine and L-arginine that have functions important
from a primarily medical viewpoint.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2011.12.033.
References
Fig. 3. Effect of pH on activities of wild-type and mutant enzymes. The reaction was
performed using Britton-Robinson buffer at a pH range of 4.0–12.0. The values are
average of two independent experiments.
the environment around the substrate binding site by mutation
affects L-histidine binding more strongly than catalytic activity.
In contrast to the L-histidine oxidation, the kcat and Km values
for L-phenylalanine oxidation were different remarkably among
the R305 variants of LGOX. The mutants that have hydrophobic
residue at the position of 305 displayed the higher kcat value for
L-phenylalanine oxidation. The mutation of R305 of LGOX with
hydrophobic amino acid might be converted to an appropriate
form for L-phenylalanine oxidation. With a focus on Km value for
L-leucine oxidation, R305A and R305L LGOX showed lower values
than R305K LGOX. The result indicates that the mutation of R305
with Ala and Leu might be converted to an appropriate form for
L-leucine binding.
From the results of our investigations, it is considered that a
subtle orientation shift of the bound substrate results from the
change of the environment around the substrate binding site by
[1] H. Kusakabe, Y. Midorikawa, T. Fujishima, A. Kuninaka, H. Yoshino, Purification
and properties of a new enzyme, L-glutamate oxidase, from Streptomyces sp. X119-6 grown on wheat bran, Agric. Biol. Chem. 47 (1983) 1323–1328.
[2] T. Kamei, K. Asano, H. Suzuki, M. Matsuzaki, S. Nakamura, L-Glutamate oxidase
from Streptomyces violascens. I. Production, isolation and some properties,
Chem. Pharm. Bull. 31 (1983) 1307–1314.
[3] A. Bohmer, A. Muller, M. Passarge, P. Liebs, H. Honeck, H.G. Muller, A novel Lglutamate oxidase from Streptomyces endus. Purification and properties, Eur. J.
Biochem. 182 (1989) 327–332.
[4] C.Y. Chen, W.T. Wu, C.J. Huang, M.H. Lin, C.K. Chang, H.J. Huang, J.M. Liao, L.Y.
Chen, Y.T. Liu, A common precursor for the three subunits of L-glutamate
oxidase encoded by gox gene from Streptomyces platensis NTU3304, Can. J.
Microbiol. 47 (2001) 269–275.
[5] S. Upadhyay, N. Ohgami, H. Kusakabe, H. Suzuki, Electrochemical
determination of c-glutamyl transpeptidase activity and its application to a
miniaturized analysis system, Biosens. Bioelectron. 21 (2006) 1230–1236.
[6] S. Upadhyay, N. Ohgami, H. Kusakabe, H. Mizuno, J. Arima, T. Tamura, K.
Inagaki, H. Suzuki, Performance characterization of recombinant L-glutamate
oxidase in a micro GOT/GPT sensing system, Sens. Actuators B: Chem. 119
(2006) 570–576.
[7] M. Nakano, T.S. Danowski, L-Amino acid oxidase (rat kidney), Methods
Enzymol. 17B (1971) 601–605.
[8] D. Wellner, L-Amino acid oxidase (snake venom), Methods Enzymol. 17B
(1971) 597–600.
T. Utsumi et al. / Biochemical and Biophysical Research Communications 417 (2012) 951–955
[9] H. Blaschko, D.B. Hope, The oxidation of L-amino acids by Mytilus edulis,
Biochem. J. 62 (1956) 335–339.
[10] P.K. Stumpf, D.E. Green, L-Amino acid oxidase of Proteus vulgaris, J. Biol. Chem.
153 (1944) 387–399.
[11] S.G. Knight, the L-amino acid oxidase of molds, J. Bacteriol. 55 (1948) 401–409.
[12] K. Burton, The L-amino-acid oxidase of Neurospora, Biochem. J. 50 (1951) 258–268.
[13] J.A. Duerre, S. Chakrabarty, L-Amino acid oxidases of Proteus rettgeri, J.
Bacteriol. 121 (1975) 656–663.
[14] H. Kusakabe, K. Kodama, A. Kuninaka, H. Yoshino, H. Misono, K. Soda, A new
antitumor enzyme, L-lysine a-oxidase from Trichoderma viride, J. Biol. Chem.
255 (1980) 976–981.
[15] H. Koyama, Purification and characterization of a novel L-phenylalanine
oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501, J.
Biochem. 92 (1982) 1235–1240.
[16] H. Koyama, Further characterization of a novel L-phenylalanine oxidase
(deaminating and decarboxylating) from Pseudomonas sp. P-501, J. Biochem.
93 (1983) 1313–1319.
[17] S. Nasu, F.D. Wicks, R.K. Gholson, L-Aspartate oxidase, a newly discovered
enzyme of Escherichia coli, is the B protein of quinolinate synthetase, J. Biol.
Chem. 257 (1982) 626–632.
955
[18] S. Kasaoka, N. Tsuboyama-Kasaoka, Y. Kawahara, S. Inoue, M. Tsuji, O. Ezaki, H.
Kato, T. Tsuchiya, H. Okuda, S. Nakajima, Histidine supplementation
suppresses food intake and fat accumulation in rats, Nutrition 20 (2004)
991–996.
[19] J. Arima, C. Sasaki, C. Sakaguchi, H. Mizuno, T. Tamura, A. Kashima, H.
Kusakabe, S. Sugio, K. Inagaki, Structural characterization of L-glutamate
oxidase from Streptomyces sp. X-119-6, FEBS J. 276 (2009) 3894–3903.
[20] R. Abagyan, M. Totrov, D. Kuznetsov, A new method for protein modelling and
design: applications to docking and structure prediction from the distorted
native conformation, J. Comp. Chem. 15 (1994) 488–506.
[21] K. Soda, Microdetermination of D-amino acids and D-amino acid oxidase
activity with 3-methyl-2-benzothiazolone hydrazone hydrochloride, Anal.
Biochem. 24 (1968) 228–235.
[22] J. Arima, T. Tamura, H. Kusakabe, M. Ashiuchi, T. Yagi, H. Tanaka, K. Inagaki,
Recombinant expression, biochemical characterization and stabilization
through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-1196, J. Biochem. 134 (2003) 805–812.
[23] P.D. Pawelek, J. Cheah, R. Coulombe, P. Macheroux, S. Ghisla, A. Vrielink, The
structure of L-amino acid oxidase reveals the substrate trajectory into an
enantiomerically conserved active site, EMBO J. 19 (2000) 4204–4215.