A new l-arginine oxidase engineered from l-glutamate oxidase

Article abstract of DOI:10.1002/pro.4070

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

Full text of DOI:10.1002/pro.4070

Received: 28 January 2021
DOI: 10.1002/pro.4070
Revised: 17 March 2021
Accepted: 21 March 2021
F U L L - L E N G T H P A P E R
A new L-arginine oxidase engineered from L-glutamate
oxidase
Yoshika Yano1 | Shinsaku Matsuo1 | Nanako Ito2 | Takashi Tamura1
|
Hitoshi Kusakabe3 | Kenji Inagaki1
| Katsumi Imada2
1Department of Biofunctional Chemistry,
Abstract
Graduate School of Environmental and
Life Science, Okayama University,
Okayama, Japan
2Department of Macromolecular Science,
Graduate School of Science, Osaka
University, Toyonaka, Osaka, Japan
3Enzyme Sensor Co., Ltd., Tsukuba,
Ibaraki, Japan
The alternation of substrate specificity expands the application range of
enzymes in industrial, medical, and pharmaceutical fields. L-Glutamate oxi-
dase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamina-
tion of L-glutamate to produce 2-ketoglutarate with ammonia and hydrogen
peroxide. LGOX shows strict substrate specificity for L-glutamate. Previous
studies on LGOX revealed that Arg305 in its active site recognizes the side
chain of L-glutamate, and replacement of Arg305 by other amino acids drasti-
cally changes the substrate specificity of LGOX. Here we demonstrate that the
R305E mutant variant of LGOX exhibits strict specificity for L-arginine. The
oxidative deamination activity of LGOX to L-arginine is higher than that of L-
arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure
analysis revealed that the guanidino group of L-arginine is recognized not only
by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305
in wild-type LGOX. Multiple interactions by these residues provide strict speci-
ficity and high activity of LGOX R305E toward L-arginine. LGOX R305E is a
thermostable and pH stable enzyme. The amount of hydrogen peroxide, which
is a byproduct of oxidative deamination of L-arginine by LGOX R305E, is pro-
portional to the concentration of L-arginine in a range from 0 to 100 μM. The
linear relationship is maintained around 1 μM of L-arginine. Thus, LGOX
R305E is suitable for the determination of L-arginine.
Correspondence
Katsumi Imada, Department of
Macromolecular Science, Graduate School
of Science, Osaka University, 1-1
Machikaneyama-cho, Toyonaka, Osaka
560-0043, Japan.
Email: kimada@chem.sci.osaka-u.ac.jp
Kenji Inagaki, Department of
Biofunctional Chemistry, Graduate School
of Environmental and Life Science,
Okayama University, Okayama, Okayama
700-8530, Japan.
Email: kinagaki@okayama-u.ac.jp
Funding information
Japan Science and Technology
Corporation, Grant/Award Number:
24560962
K E Y W O R D S
crystal structure, L-arginine oxidase, L-glutamate oxidase, modification of substrate specificity,
substrate recognition
1 | INTRODUCTION
L-Arginine is a semi-essential amino acid for humans and
is involved in various metabolic pathways to make prod-
ucts of biological importance, including nitric oxide,
agmatine, creatine, citrulline, ornithine, and poly-
amines.1,2 The cardiovascular system and the nerve sys-
tem are affected by L-arginine through the production of
Abbreviations: AROD, L-arginine oxidase; LAAO, L-amino acid
oxidase; LGOX, L-glutamate oxidase.
Yoshika Yano, Shinsaku Matsuo, and Nanako Ito contributed equally to
this work.
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© 2021 The Protein Society.
wileyonlinelibrary.com/journal/pro
Protein Science. 2021;30:1044–1055.

YANO ET AL.
1045
nitric oxide.3,4 L-Arginine can be a medical biomarker
because patients with argininemia or cancer show
unusual concentrations of L-arginine in the blood.5,6
Thus, various enzymatic methods have been developed
for quick and easy determination of L-arginine levels. The
method using arginase (E.C. 3.5.3.1) and urease (E.C.
3.5.1.5) detects the ammonia produced from L-arginine
by the two coupling reactions: arginase degrades L-
arginine to urea and L-ornithine, and urease produces
ammonia from urea.7,8 The L-arginine decarboxylase
(EC 4.1.1.19) method detects the carbonate ion, which is
produced by the decarboxylation of L-arginine, by ion
selective membrane electrode.9 The method using L-
arginine deiminase (EC 3.5.3.6) and argininosuccinate
synthetase (EC 6.3.4.5) measures the pyrophosphate pro-
duced from L-arginine thorough the two-step reaction.
Pyrophosphate is a byproduct of the argininosuccinate
synthesis from L-citrulline by argininosuccinate synthe-
tase, and L-citrulline is converted from L-arginine by L-
arginine deiminase.10 However, these methods need com-
plicated multiple enzymatic reactions or special elec-
trodes, and therefore their application is limited.
Recently, Matsi et al. purified L-arginine oxidase (AROD,
EC 1.4.3.25), which is a flavoenzyme that catalyzes the
oxidative deamination of L-arginine to 2-ketoarginine,
from Pseudomonas sp. TPU 7192 and demonstrated that
AROD can be used for the determination of L-arginine.11
However, the specific activity to L-arginine of AROD is
rather low (0.19 U mg−1). Thus, a new enzymatic method
is desired for L-arginine determination.
Therefore, LGOX has been used as a biosensor to quan-
tify L-glutamate and been applied in quality management
of foods and fermentation processes. LGOX can also
detect glutamic oxaloacetic transaminase, glutamic
pyruvic transaminase and γ-glutamyl transpeptidase
activities, which are diagnostic markers of liver func-
tion.19,20 Thus, LGOX is used in biosensors for clinical
testing apparatus. We determined the crystal structure of
LGOX and found that Arg305 is located in the potential
substrate binding site of LGOX.21 The following mutation
study based on the structure and a substrate-docking sim-
ulation revealed that Arg305 plays a key role in the recog-
nition of the side chain of L-glutamate.22 Interestingly,
point mutation of Arg305 drastically changed the sub-
strate specificity of LGOX. R305L, R305A, and R305K
mutant variants of LGOX lost the activity to L-glutamate,
but gained an activity to L-histidine. LGOX R305D
showed an activity to L-arginine although the activity to
L-glutamate was completely lost.22 These results suggest
that various LAAOs specific to certain L-amino acids can
be developed by mutation of LGOX.
Here we show that the R305E mutant variant of
LGOX exhibits strict specificity for L-arginine. The spe-
cific activity of LGOX R305E to L-arginine is much higher
than that of AROD and that of LGOX R305D. The crystal
structures of LGOX R305E and its complex with L-argi-
nine revealed that the molecular mechanism of strict
specificity for L-arginine. These findings indicate that
LGOX R305E is a useful enzyme for determination of L-
arginine.
L-Amino acid oxidases (LAAO) are widely distributed
flavoenzymes that catalyze the oxidative deamination of
an L-amino acid to produce a 2-oxo acid with hydrogen
peroxide and ammonia as byproducts.12 LAAOs are
involved in various biological and physiological functions
in many organisms, including venomous snakes, mam-
mals, insects, fish, mollusks, fungi, and bacteria.13 While
many LAAOs exert broad substrate specificity, several
LAAOs, such as L-glutamate oxidase from Streptomyces
sp. X-119-6 (LGOX; EC 1.4.3.11),14 L-lysine-α-oxidase
from Trichoderma viride (LysOX)15,16 and L-phenylala-
nine oxidase from Pseudomonas sp. P-501 (PAO),17,18
show strict substrate specificity.
LGOX is a member of the LAAO family and catalyzes
the oxidative deamination of the α-amino group of L-glu-
tamate to 2-ketoglutarate via an imino acid intermediate.
LGOX is produced as a single polypeptide precursor of
701 residues and is processed into three polypeptide
chains to exert the enzymatic activity. The N-terminal
14 residues, the C-terminal 18 residues, and 40 residues
between the second and the third chains are removed in
the mature enzyme. LGOX shows strict substrate specific-
ity for L-glutamate, and high thermal and pH stability.14
2 | RESULTS
2.1 | LGOX R305E shows strict specificity
toward L-arginine
LGOX R305E was prepared by site-directed mutagenesis.
LGOX R305E was expressed in E. coli JM109 and was
purified by two-step column chromatography after matu-
ration using
a metalloprotease from Streptomyces
griseus21 (Table 1). We obtained 13 mg of purified
enzyme from 7 g of cell pellet. The purified protein
showed a single band in Native-PAGE analysis (Figure 1
(a)) and three bands (relative molecular weights of 40, 17,
and 10 kDa) corresponding to α, β, and γ-fragments in
SDS-PAGE analysis (Figure 1(b)), indicating that mature
LGOX R305E was successfully purified. The final purified
enzyme exhibited specific activity of 7.5 U mg−1
(Table 2).
Oxidative deamination activity of LGOX R305E for
various L-amino acids were assessed using the
4-aminoantipyrine phenol method.21 We tested 23 L-

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YANO ET AL.
TABLE 1 Summary of purification of LGOX R305E
Total
protein (mg)
Total
activity (U)
Specific
activity (Umg−1
Yield
(%)
Purification
(fold)
)
Crude extract
937
125
76
59
553
184
0.063
4.4
100
936
311
1
Protease digestion
70
Ammonium sulfate (30%–
2.4
38
60%)
DEAE Toyopearl 650 M
Sephacryl S 300 HR
19
13
110
99
5.7
7.5
186
168
91
118
Note: The activity of LGOX R305E was measured at 40ꢀC, pH 8.0.
FIGURE 1 LGOX R305E shows
strict specificity for L-arginine. (a) Blue
native-PAGE analysis of LGOX R305E.
In 10 μg of protein was applied. (b) SDS-
PAGE analysis of purified LGOX R305E:
lane M, molecular mass marker; lane
1, purified LGOX R305E.
(c) Comparison of substrate specificity
profiles of LGOX R305E and wild-type
LGOX. Relative oxidative activities of
LGOX R305E and wild-type LGOX are
indicated in red, and blue bars,
respectively. The substrate that showed
the highest activity was set as 100%
relative activity. Enzyme activities were
measured using 1 mM substrates at 40ꢀC
TABLE 2 Kinetic parameters of LGOX, LGOX R305E, R305D, and AROD
Substrate
L-Arg
Specific activity (Umg−1
)
Km (mM)
0.223 0.071
7.20
kcat (s−1
)
kcat/Km (M−1 s−1
1.53 × 104
2.29 × 102
3.08 × 105
-
)
LGOX R305E
LGOX R305D22
LGOX22
7.5
1.5
3.42 0.61
1.65
L-Arg
L-Glu
57
0.173
53.2
AROD11
L-Arg
0.19
0.149
-
Note: Kinetic parameters of LGOX R305E was measured at 40ꢀC, pH 8.0. Each data represents the mean SD of triplicate measurements.

YANO ET AL.
1047
α-amino acids and found that LGOX R305E exhibits the
highest activity toward L-arginine and low activity toward
L-tyrosine, L-histidine, L-lysine, and L-ornithine with rela-
tive activities of 3.7% 0.29, 3.5% 0.11, 3.4% 0.25,
and 2.4% 0.12 of L-arginine, respectively. No activity
was observed for the other L-α-amino acids including the
original substrate, L-glutamate. Therefore, the R305E
mutation changes the substrate specificity of LGOX from
L-glutamate to L-arginine.
original substrate to the wild-type enzyme. In contrast,
the catalytic efficiency (kcat/Km) of LGOX R305E for
L-arginine was 5% of that of LGOX for L-glutamate.
2.3 | The effect of pH and temperature
on the enzymatic activity of LGOX R305E
We next characterized the enzyme activity of LGOX
R305E toward L-arginine by monitoring the hydrogen
peroxide production using the 4-aminoantipyrine phenol
method.21 LGOX R305E exhibited the highest specific
activity of 7.5 U mg−1 for L-arginine at pH 8 and 40ꢀC.
This value is 40-fold higher than that of AROX from
Pseudomonas sp. TPU 7192 (0.19 U mg−1).11
The enzyme activity was retained more than 90% of
the highest activity from 30ꢀC to 50ꢀC at pH 7.4 and was
dropped to 75% at 60ꢀC (Figure 2(a)). The remaining
activity was 100% after 1 h incubation at 50ꢀC and 88% at
60ꢀC. The enzyme still showed 60% remaining activity
after 1 h incubation at 70ꢀC (Figure 2(b)). The melting
2.2 | Kinetic properties of LGOX R305E
The kinetic parameters of LGOX R305E for L-arginine
under the optimum condition (pH 8 and 40ꢀC) were
determined by the 4-aminoantipyrine phenol method
and are summarized in Table 2. The Km value of LGOX
R305E for L-arginine (0.22 mM) is close to that of LGOX
for L-glutamte, (0.17 mM) and that of AROD for L-argi-
nine (0.15 mM), indicating that the binding affinity of L-
arginine to LGOX R305E is comparable to that of the
FIGURE 2 Comparison of enzyme properties of LGOX R305E with wild-type LGOX. (a) Temperature dependence of the enzyme
activities of LGOX R305E (closed circle) and wild-type LGOX (open circle). Relative activities at pH 7.4 from 20ꢀC to 70ꢀC are plotted. The
maximal activity is set as 100% relative activity (40ꢀC for LGOX R305E and 50ꢀC for wild-type LGOX). (b) The remaining activities after
incubation at various temperatures (30–70ꢀC) for 1 h. Relative activities of LGOX R305E (closed circle) and wild-type LGOX (open circle) are
plotted. The activity without heat treatment was set as 100% remaining activity. (c) Far-UV CD curves of LGOX R305E (black) and wild-type
LGOX (gray) at 210 nm obtained by temperature scan from 25ꢀC to 90ꢀC. (d) Effect of pH on the activity of LGOX R305E (closed circle) and
wild-type LGOX (open circle). The activity at optimum pH was set as 100% relative activity. (d) The remaining activities after incubation at
various pH for 1 h on ice on stability. Relative activities of LGOX R305E (closed circle) and wild-type LGOX (open circle) are plotted. The
activity at pH 7.4 was set as 100% remaining activity

1048
YANO ET AL.
temperature (Tm) estimated from circular dichroism
(CD) data at 212 nm was 71ꢀC for LGOX R305E and
74ꢀC for the wildtype enzyme (Figure 2(c)). Therefore,
LGOX R305E is a thermostable enzyme, although it is
slightly thermally unstable compared with wild-type
LGOX (Figure 2(a), (b)).
The enzyme activity was kept more than 80% of the
highest activity in the pH range from 8.0 to 9.0 at 40ꢀC
and was reduced to about 15% of the maximum at pH 6
and 35% at pH 10 (Figure 2(c)). The residual activity was
over 80% in the pH range of 5.0–11.0 (Figure 2(d)). Thus,
LGOX R305E is pH stable, comparable to the wild-
type LGOX.
FIGURE 3 Linear relationship between hydrogen peroxide
production by LGOX R305E and L-arginine concentration. The
absorbance at 555 nm is plotted against the L-arginine
concentration from 0 to100 μM. The magnified plot in the range
from 0 to 10 μM of L-arginine is shown as an inset. The linear
regression equation with the coefficient of determination is also
shown in the graph
2.4 | Determination of L-arginine
concentration using LGOX R305E
LGOX R305E shows strict substrate specificity for L-argi-
nine, and its enzyme activity on L-arginine is much
higher than that of AROD. Oxidative deamination of L-
arginine by LGOX R305E produces hydrogen peroxide,
which can easily be detected. Thus, LGOX R305E is
expected to be useful for determining the concentration
of L-arginine. We prepared solutions with various con-
centrations of L-arginine (0–100 μM) and tested LGOX
R305E to measure the concentration of L-arginine using
the 4-aminoantipyrine-TOOS (N-ethyl-N-[2-hydroxy-
3-sulfopropyl]-m-toluidine) method, which quantifies
hydrogen peroxide.23 Each L-arginine solution was
added to the reaction mixture containing 0.25 U ml−1 of
LGOX R305E. Then, we monitored absorbance at
555 nm at 28ꢀC and found that the absorbance reached
a maximum after around 5 min incubation. The maxi-
mum values were recorded and plotted against the L-
arginine concentration. The plots indicated a linear rela-
tionship between the 555 nm absorbance and L-arginine
concentration in a range from 0 to 100 μM of L-arginine
concentration with a coefficient of determination of
0.9993 (Figure 3). The plot also showed liner relation-
ship between 0 and 10 μM of L-arginine, indicating that
LGOX R305E is potentially useable to determine L-argi-
nine concentration.
modeled, because these residues did not appear in the
electron density map.
The LGOX R305E crystal belongs to the space group
of P6122, which is the same space group as LGOX, with
cell dimensions of a = b = 124.0 and c = 168.6 Å, which
are similar to those of the LGOX crystal.21 A single
LGOX R305E molecule exists in an asymmetric unit and
forms a dimer with a neighboring molecule related by
crystallographic two-fold symmetry (Figure 4(a)), as
seen in the LGOX crystal. The structure of LGOX R305E
is almost identical to that of LGOX. LGOX R305E con-
sists of three domains: the FAD-binding domain (E17–
G96, G332–T430, and Y621–A671), the substrate-
binding domain (R97–R204, A324–G331, and H431–
V620) and the helical domain (R205–R323) (Figure 4
(b)). No significant structural difference was observed
between LGOX R305E and LGOX except for the loop
region (F314–T325) connecting the helical domain with
the substrate binding domain (Figure 4(c), (d)). The loop
of LGOX R305E overhangs the substrate binding pocket,
and the guanidino group of R317 in the loop lies above
the isoalloxazine ring of FAD and forms a cation-π inter-
action with FAD (Figure 4(d)). Therefore, a large con-
formational change of the loop is needed to place a
substrate in the active site of LGOX R305E. The loop
conformation is further stabilized by the interaction
between the carboxy group of E305 and the hydroxyl
group of S318. However, it is unclear how the R305E
mutation induces the conformational change of the loop
from the wild-type conformation.
2.5 | Crystal structure of LGOX R305E
The structure of LGOX R305E was determined at 2.65 Å
resolution (Figure 4(a), Table 3). We crystallized LGOX
R305E composed of three fragments, α (residue 15–390),
γ (391–480), and β (521–683). However, residues 15–16,
363–371, 387–390, 471–480, 521, and 672–683 were not

YANO ET AL.
1049
FIGURE 4 Structure of LGOX R305E. (a) Ribbon representation of the LGOX R305E dimer. One subunit is colored in rainbow from the
N-terminus (blue) to the C-terminus (red), and the other in pink. (b) Cα trace of a single subunit of LGOX R305E. The domain structure is
depicted in different colors: cyan, the FAD-binding domain; yellow, substrate-binding domain; green, helical domain. FAD is shown in the
stick model colored by element: red, oxygen; blue, nitrogen; gray, carbon. The entrance of the funnel is shown by an arrow.
(c) Superposition of Cα trace of wild-type LGOX (pink), LGOX R305E (cyan), and LGOX R305E in complex with L-arginine (yellow). The
residues S313-T325 (in the black box) are highlighted in different colors: wild-type LGOX, pale blue; LGOX R305E, violet; LGOX R305E in
complex with L-arginine, orange. (d) Close-up view of the loop connecting the helical domain with the substrate binding domain. The
residues S313-T325 are colored different from the rest. Wild-type LGOX, LGOX R305E, and LGOX R305E in complex with L-arginine are
shown in the left, middle and right panels, respectively. The colors are the same as (c)
2.6 | The structural basis of L-arginine
recognition by LGOX R305E
with its indole ring of W653. The α-carboxy group of L-
arginine interacts with R124 and Y562. These residues
are conserved in other LAAOs.12,24,25 The side chain of L-
arginine is recognized by E305, D433, W564, and E617
(Figure 5(a)). The guanidino group stacks on the indole
ring of W564 to form a cation-π interaction and forms
hydrogen-bonds with the side chain carboxy groups of
E305, D433, and E617. These multiple interactions con-
tribute to the specific binding of L-arginine to the active
site of LGOX R305E.
The side chain of W371 changes the conformation by
substrate binding and blocks the path to the active site.24
Similar side chain movement by L-arginine binding was
observed in LGOX R305E. However, the path to the
active site of LGOX R305E is much wider than LysOX,
and therefore, the active site of LGOX R305E is still
accessible after L-arginine binding.
The crystal of LGOX R305E in complex with L-arginine
was prepared by soaking method. The color of the crys-
tals was changed from yellow to colorless after 5–
10 min soaking in L-arginine solution, suggesting that
FAD is converted to the reduced form and that the sub-
strate is bound to the enzyme in the crystal.24,25 We
collected X-ray diffraction data from the colorless
crystals.
In the substrate-complex structure, the loop between
the helical and the substrate-binding domains moves
away from the substrate binding pocket (Figure 4(d)),
and L-arginine lies on the isoalloxazine ring of FAD. The
amino acid backbone of L-arginine is recognized by
LGOX R305E in the same manner as other LAAOs
(Figure 5(a)), such as LysOX (Figure 5(b))24 and LAAO
from Calloselasma rhodostoma venom (CrLAAO).25 The
α-amino group of L-arginine forms hydrogen bonds with
the carbonyl oxygen of A652 and a cation-π interaction
R124, E305, and W564 adopt alternative conforma-
tions in the substrate complex structure. This is probably
because the substrate does not bind in all molecules in
the crystal. In fact, the refined occupancy of the bound L-

1050
YANO ET AL.
TABLE 3 Data collection and
refinement statistics
LGOX R305E
LGOX R305E-L-Arg
Data collection
Space group
Cell dimensions
a, b, c (Å)
P6122
P6122
124.0, 124.0, 168.6
90, 90, 120
124.8, 124.8, 170.2
90, 90, 120
α, β, γ (ꢀ)
Resolution (Å)
Rpim
84.3–2.65 (2.78–2.65)
0.034 (0.191)
18.5 (4.6)
54.0–2.7 (2.83–2.7)
0.089 (0.317)
7.7 (2.5)
I / σI
Completeness (%)
Redundancy
Refinement
Resolution (Å)
No. reflections
Rwork / Rfree
No. atoms
99.9 (100)
96.4 (99.1)
12.3 (13)
4.8 (4.8)
66.3–2.65 (2.77–2.65)
22,853 (2,774)
54.0–2.7 (2.82–2.70)
21,093 (2,660)
19.1/24.1 (21.6/29.7)
19.8/25.5 (25.9/35.4)
Protein
4,723
53
4,800
65
Ligand/ion
Water
87
190
B-factors
Protein
45.4
29.4
40.1
30.4
28.1
30.5
Ligand/ion
Water
R.m.s. deviations
Bond lengths (Å)
Bond angles (ꢀ)
Ramachandran plot (%)
Preferred regions
Allowed regions
Outliers
0.006
0.860
0.002
0.549
95.2
4.5
97.0
3.0
0
0.3
Note: Values in parentheses are for highest-resolution shell.
arginine is 0.78. Substrate binding may induce conforma-
tional change of these residues.
glutamate. The crystal structures revealed that the molec-
ular mechanism of the substrate specificity change and
the structural basis of the high affinity for L-arginine
(Figure 6). The substrate specificity change is not just the
result of simple replacement of a basic residue with an
acidic residue in the active site. The guanidino group of
L-arginine does not only interact with E305 but also with
D433, W564, and E617, which are conserved in wild-type
LGOX (Figure 6(a), (b)). D433 and E617 are trapped by
R305 and the negative charge of these residues are neu-
tralized by R305 in the wild-type LGOX structure
(Figure 6(a)). Moreover, R305 sterically blocks W564 to
interact with the substrate in LGOX. The R305E
3 | DISCUSSION
Previous structural and mutational studies revealed that
Arg305 is important for substrate recognition of LGOX
and demonstrated that point mutation at Arg305 can
drastically change the substrate specificity of LGOX.21,22
Here we found that the R305E mutant variant of LGOX
shows strict specificity for L-arginine. The Km value for L-
arginine is almost the same level as that of LGOX for L-

YANO ET AL.
1051
FIGURE 5 Comparison of the substrate-binding site structure of LGOX R305E with that of LysOX. Stereo view of the substrate binding
structure of LysOX-Lys (a), and LGOX R305E-Arg (b). The substrate molecules are colored in green. The protein residues and FAD are
shown in yellow for LysOX and cyan for CrLAAO. The nitrogen and oxygen atoms are colored in blue and red, respectively. The bound
water molecules are shown as red balls. Possible hydrogen bonds and cation-π interactions are indicated by red broken lines
mutation releases D433, W564, and E617 and allows
them to interact with the substrate bound in the active
site of LGOX R305E. These multiple interactions by these
residues as well as E305 provide high affinity for L-argi-
nine. In addition, R305E mutation makes the charge of
the active site surface highly negative with the contribu-
tion of D433 and E617. This charge distribution attracts
the basic amino acids but repels the acidic amino acids
from the active site. Due to the charge distribution,
LGOX R305E shows low enzymatic activity toward the
basic amino acids, such as L-histidine, L-lysine, and L-
ornithine, while wild-type LGOX does not at all.
found in other LAAOs that bind hydrophobic amino
acids.25–27 However, LGOX R305E has no such hydro-
phobic hole (Figure 5(a)). E617 is present in the
corresponding position of F439 of LysOX. No residue
exists in the corresponding position of F216 of LysOX.
Therefore, LGOX R305E does not bind hydrophobic
amino acids (Figure 5).
We previously showed that LGOX R305D has activity
toward L-arginine (1.5 U mg−1),22 but the specific activity
is 20% of that of LGOX R305E. The Km value of LGOX
R305E for L-arginine is approximately 3% of that of
LGOX R305D, indicating that LGOX R305E has higher
LysOX recognizes the hydrophobic side chain arm of
the substrate L-lysine by hydrophobic hole composed of
five hydrophobic residues (F216, W371, F439, A475, and
W476)24 (Figure 5(b)). The hydrophobic hole is also
substrate-binding affinity. The catalytic efficiency (kcat/
Km) is approximately 65-fold higher than that of LGOX
R305D. The structure of the L-arginine complex of LGOX
R305E suggests that the side chain of D305 is too short to

1052
YANO ET AL.
FIGURE 6 Comparison of the active site structure of L-arginine complex of LGOX R305E with that of wild-type LGOX. Active site of
ligand-free wild-type LGOX (a) and of LGOX R305E with L-arginine (b). The nitrogen and oxygen atoms are colored in blue and red,
respectively. Possible hydrogen bonds are shown as red, dashed lines. (c) Superposition of the active site of LGOX R305E (green) and LGOX
R305E in complex with L-arginine (cyan)
interact with the guanidino group of L-arginine in the
active site and therefore the side chain of the L-arginine
may be recognized only by D433, W564, and E617 in
LGOX R305D. Interactions with D433, W564, and E617
may not be enough to bind the L-arginine in proper orien-
tation for the reaction. Thus, the activity of LGOX R305D
toward L-arginine is weaker than that of LGOX R305E.
In this study, we showed that LGOX R305E has a
strict substrate specificity for L-arginine. The specific
activity of LGOX R305E to L-arginine is much higher
than that of native AROD. The thermal and pH stability
of LGOX R305E is almost the same level as wild-type
LGOX, which has been widely used for L-glutamate sens-
ing. These properties are suitable for application to an
enzyme-based sensor. Hydrogen peroxide is produced as
a byproduct of oxidative deamination of L-arginine by
LGOX R305E. We demonstrated that the amount of
hydrogen peroxide detected by the 4-aminoantipyrine-
TOOS method is proportional to the concentration of L-
arginine from 0 to 100 μM. Moreover, the linear relation-
ship is maintained even around 1 μM of L-arginine. These
results indicate that LGOX R305E based method is a
powerful tool to determine L-arginine.
(Takara, Japan), plasmid pGOx_mal1 as a template, and
the following oligonucleotide primers: 50-CATGACCTC
GGAACTCCACCTGC-30, and 50-CAGGTGGAGTTCCG
AGGTCATG-30. The template plasmid was digested with
Dpn I at 37ꢀC for 1 h. The plasmid carrying LGOX R305E
(pGOx_mal1_305E) was transformed into E. coli JM109.
The introduction of the mutation was confirmed by DNA
sequencing.
4.2 | Purification of LGOX R305E
E. coli JM109 harboring pGOx_mal1_305E was cultured
at 22ꢀC for 24 h in 1 L of 2 × YT medium containing
50 μg ml−1 ampicillin. Expression of the LGOX R305E
gene was induced by the addition of 0.1 mM IPTG for
22 h at 22ꢀC. Cells were harvested by centrifugation and
stored at −80ꢀC until use. The pellet was thawed and
resuspended in 20 mM potassium phosphate buffer
(KPB) at pH 7.4 and sonicated at 150 W for 15 min at
4ꢀC. After centrifugation, the supernatant was stored
with metalloprotease from Streptomyses griseus (Sigma-
Aldrich Corp., St. Louis, MO, USA) at 30ꢀC for 18 h for
maturation22 and then was incubated at 60ꢀC for 20 min
to inactivate the protease. The solution was centrifuged,
and the supernatant was brought to 30% saturation with
ammonium sulfate for 30 min on ice. After removal of
the precipitate, the supernatant was brought to 60% satu-
ration with ammonium sulfate for 30 min on ice. The
precipitate was collected by centrifugation, dissolved in
20 mM KPB (pH 7.4), and dialyzed for 16 h against
20 mM KPB (pH 7.4) with 100 mM NaCl. The solution
4 | METHODS
4.1 | Site-directed mutagenesis
The expression vector of LGOX (pGOx_mal1) was con-
structed as described previously.22 Site-directed mutagenesis
was conducted by inverse PCR using KOD Plus Neo

YANO ET AL.
1053
was loaded on a DEAE Toyopearl 650 M column (Tosoh
Corp., Tokyo, Japan) equilibrated with 100 mM NaCl in
20 mM KPB (pH 7.4) and was eluted with 20 mM KPB
(pH 7.4) containing 200 mM NaCl. The fractions con-
taining LGOX R305E were concentrated and applied to a
Sephacryl S 300 HR column (GE healthcare). The peak
fractions were collected and pooled. The protein concen-
tration was determined by the Bradford method using a
Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) with bovine serum albumin as a
standard. Wild-type LGOX was expressed and purified as
previously described.22 The purity of the enzyme was
assessed by SDS-PAGE (15%) and Native-PAGE (7.5%).
316 nm by Shimadzu UVmini-1,240 UV–VIS spectropho-
tometer. One unit of activity was taken as the amount of
enzyme that liberates 1 μmol of α-keto acid per minute.
4.4 | Determination of substrate
specificity
The substrate specificity of LGOX R305E and wild-type
LGOX was determined by measuring enzyme activities
for 23 L-α-amino acids using the 4-aminoantipyrine phe-
nol method.
4.5 | Determination of optimum pH and
temperature
4.3 | Enzyme assay
The enzyme activity of LGOX R305E was measured by
detecting hydrogen peroxide using the 4-aminoantipyrine
phenol method or by monitoring α-keto acid using the
3-methyl-2-benzothiazolinone hydrazone (MBTH) method.
The enzyme assay by the 4-aminoantipyrine phenol
method was conducted as described previously with
slight modification.28 0.9 ml of reaction mixture con-
taining 50 mM buffer (borate-NaOH buffer (pH 8.0) for
LGOX R305E and KPB (pH 7.4) for wild-type LGOX),
2 mM phenol, 2 mM 4-aminoantipyrine, 10 U ml−1
horseradish peroxidase, and an appropriate amount of
enzyme was pre-incubated at 40ꢀC for 3 min. The reac-
tion was initiated by the addition of 0.1 ml of 100 mM
substrate solution, and the formation of red quinoimine
dye was monitored at a wavelength of 505 nm by a
Shimadzu UVmini-1,240 UV–VIS spectrophotometer
(Shimadzu GLC Ltd., Tokyo, Japan). One unit of activity
was defined as the amount of enzyme that catalyzes the
formation of 1 μmol of hydrogen peroxide per minute.
The enzyme assay by the MBTH method was per-
formed as previously described with slight modification.29
0.9 ml of reaction mixture containing 70 mM buffer
(borate-NaOH buffer (pH 8.0) for LGOX R305E and KPB
(pH 7.4) for wild-type LGOX), 300 U ml−1 catalase, and
an appropriate amount of enzyme was pre-incubated at
40ꢀC for 3 min. The reaction was started by the addition
of 0.1 ml of 100 mM L-arginine for LGOX R305E and
10 mM L-glutamate for wild-type LGOX followed by incu-
bation at 40ꢀC for 5 min. The reaction was stopped by
adding 0.1 ml of 25% trichloroacetic acid to the reaction
mixture. Then 1.9 ml of 1 M acetate buffer (pH 5.0) and
0.8 ml of 1 mg ml−1 MBTH aqueous solution were added
to the reaction mixture, and the mixture was incubated
at 50ꢀC for 30 min. After cooling the mixture at room
temperature for 20 min, the formation of an MBTH-azine
α-keto acid derivative was measured at a wavelength of
Optimum pH was determined by measuring enzyme
activity (LGOX R305E for L-arginine at 40ꢀC; wild-type
LGOX for L-glutamate at 50ꢀC) using the
4-aminoantipyrine method. The pH was adjusted using a
universal buffer (50 mM boric acid, 50 mM citric acid,
50 mM acetic acid, 50 mM NaH2PO4, 50 mM CAPS, and
25 mM HEPES) by adding 5 mol L−1 NaOH solution. The
optimum temperature was determined by measuring
enzyme activity (LGOX R305E for L-arginine; wild-type
LGOX for L-glutamate) in 70 mM KPB (pH 7.4) using the
MBTH method.
4.6 | Measurement of residual activity
To determine the thermostability, enzyme activity
(LGOX R305E for L-arginine; wild-type LGOX for L-gluta-
mate) was measured by the MBTH method at 40ꢀC after
incubation of the enzyme in KPB (pH 7.4) at various tem-
peratures (30–90ꢀC) for 1 h. To evaluate the pH stability
of the enzyme, enzyme activity (LGOX R305E for L-argi-
nine; wild-type LGOX for L-glutamate) was measured by
the 4-aminoantipyrine phenol method at 40ꢀC for LGOX
R305E and at 50ꢀC for wild-type LGOX after incubation
of the enzyme in universal buffer at various pH (pH 3.0–
12.0) for 1 h on ice. The pH value of the enzyme solution
was adjusted to pH 7.4 by adding 100 mM KPB before
the enzyme activity assay.
4.7 | Circular dichroism spectroscopy
Far-UV CD data were collected on Jasco-720 W spectro-
polarimeter with a Peltier type cell holder for temperature
control (JASCO International Co., Tokyo, Japan). A quartz
cell with 1 mm path-length were filled with 0.33 mg ml−1

1054
YANO ET AL.
of protein solutions containing 20 mM sodium phosphate
(pH 7.4) and 150 mM NaCl. CD melting curves were mea-
sured at 210 nm over the temperature rage 25ꢀC-90ꢀC at a
heating rate of 1ꢀC min−1 with data acquisition interval of
0.2ꢀC, response time 8 s and bandwidth 2 nm.
crystals were prepared by soaking technique. The crystals
were soaked in a reservoir solution containing 10% (v/v)
MPD and 35 mM L-arginine and were stored at 277 K
until the yellow color disappeared (typically 5–10 min).
4.11 | Data collection and structure
determination
4.8 | Determination of the kinetic
parameters
The X-ray data were corrected at SPring-8 beamlines,
BL41XU and BL26B1 (Harima, Japan), with the approval
of the Japan Synchrotron Radiation Research Institute
(JASRI) (Proposal No. 2016B2719, 2017A2585, and
2017B2585). The crystals were transferred into liquid
nitrogen for freezing. The R305E variant crystals were
soaked in a cryo-protectant solution containing 10% (v/v)
MPD and 90% (v/v) of the reservoir solution for several
seconds before freezing. The diffraction data were col-
lected under nitrogen gas flow at 100 K. The diffraction
data were indexed and integrated with MOSFLM,30 and
were scaled with AIMLESS.31 The diffraction data statis-
tics are summarized in Table 3. The initial phase was
obtained by the molecular replacement method with
Phaser32 using the structure of wild type LGOX (PDB:
2E1M) as a search model. The atomic models were built
with Coot33 and refined with PHENIX.34 The structural
refinement statistics are shown in Table 3.
Enzyme activity for L-arginine with different concentra-
tions was measured at 40ꢀC using the 4-aminoantipyrine
phenol method. Kinetic parameters were determined by
Lineweaver–Burk plot.
4.9 | L-arginine determination using
LGOX R305E
The amount of hydrogen peroxide produced by oxidative
deamination of L-arginine by LGOX R305E was measured
using 4-aminoantipyrine-TOOS (N-ethyl-N-[2-hydroxy-
3-sulfopropyl]-m-toluidine) method.23 0.9 ml of reaction
mixture containing 20 mM Tris–HCl buffer (pH 9.0),
0.4 mM TOOS (N-ethyl-N-[2-hydroxy-3-sulfopropyl]-m-tolu-
idine), 0.4 mM 4-aminoantipyrine, 10 U ml−1 horseradish
peroxidase, and 0.25 U ml−1 was pre-incubated at 28ꢀC for
3 min. The reaction was started by adding 0.1 ml of L-argi-
nine solution (0–1 mM) at 28ꢀC, and the formation of qui-
none diamide was monitored at a wavelength of 555 nm for
10 min by Eppendorf Biospectrometer kinetic (Eppendorf
Co., Ltd., Tokyo, Japan). The maximum values were
recorded and plotted against the L-arginine concentration.
ACKNOWLEDGEMENTS
We thank SPring-8 beamline staffs for technical help in
use of beamline BL26B1 and BL41XU, Ayaka Yamaguchi
and Analytical Instrument Faculty of Graduate School of
Science, Osaka University for supporting CD measure-
ments. This work was supported by JSPS KAKENHI
Grant Numbers 24560962 (to Kenji Inagaki).
4.10 | Preparation of crystals
AUTHOR CONTRIBUTIONS
Initial crystallization screening was performed at 277 K
and 293 K by the sitting-drop vapor diffusion method
using the following screening kit: Wizard I and II, Cryo I
and II (Emerald Biostructures) and Crystal Screen I and
II (Hampton Research). Each drop was prepared by
mixing 1 μL protein solution (8.7 mgꢁml−1) with an equal
volume of reservoir solution. Clusters of small crystals
were grown from the condition containing 100 mM Tris–
HCl (pH 7.0), PEG8000, and MgCl2. The crystallization
conditions were optimized by screening additives and the
concentration of the precipitants and additives. The best
crystals were grown at 277 K from drops prepared by
mixing protein solution (8.7 mgꢁml−1) containing 20 mM
KPB (pH 7.4) with an equivalent volume of reservoir
solution containing 100 mM Tris–HCl (pH 7.0), 12% (v/v)
PEG8000, and 75 mM MgCl2. The substrate complex
Yoshika Yano: Investigation; writing-original draft.
Shinsaku Matsuo: Investigation. Nanako Ito: Investi-
gation; visualization. Takashi Tamura: Investigation.
Hitoshi Kusakabe: Resources. Kenji Inagaki: Concep-
tualization; project administration; resources; supervi-
sion; writing-original draft; writing-review & editing.
Katsumi Imada: Conceptualization; investigation; pro-
ject administration; supervision; visualization; writing-
original draft; writing-review & editing.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Kenji Inagaki https://orcid.org/0000-0003-3795-0184
Katsumi Imada https://orcid.org/0000-0003-1342-8885

YANO ET AL.
1055
20. Upadhyay S, Ohgami N, Kusakabe H, Suzuki H. Electrochemi-
cal determination of gamma-glutamyl transpeptidase activity
and its application to a miniaturized analysis system. Biosens
Bioelectron. 2006;21:1230–1236.
REFERENCES
1. Grillo MA, Colombatto S. Arginine revisited. Amino Acids.
2004;26:345–351.
2. Morris SM Jr. Enzymes of arginine metabolism. J Nutr. 2004;
21. Arima J, Sasaki C, Sakaguchi C, et al. Structural characteriza-
tion of L-glutamate oxidase from Streptomyces sp. X-119-6.
FEBS J. 2009;276:3894–3903.
134:2743S–2747S.
3. Wu G, Meininger CJ. Arginine nutrition and cardiovascular
function. J Nutr. 2000;130:2626–2629.
22. Utsumi T, Arima J, Sakaguchi C, et al. Arg305 of Streptomyces
L-glutamate oxidase plays a crucial role for substrate recogni-
tion. Biochem Biophys Res Commun. 2012;417:951–955.
23. Tamaoku K, Ueno K, Akiura K, Ohkura Y. New water-soluble
hydrogen donors for the enzymatic photometric determination
of hydrogen peroxide. II. N-ethyl-N-(2-hydroxy-3-sulfopropyl)
aniline derivatives. Chem Pharm Bull. 1982;30:2492–2497.
24. Kondo H, Kitagawa M, Matsumoto Y, et al. Structural basis of
strict substrate recognition of L-lysine α-oxidase from
Trichoderma viride. Protein Sci. 2020;29:2213–2225.
25. Moustafa IM, Foster S, Lyubimov AY, Vrielink A. Crystal struc-
ture of LAAO from Calloselasma rhodostoma with an L-
phenylalanine substrate: Insights into structure and mecha-
nism. J Mol Biol. 2006;364:991–1002.
26. Zhang H, Teng M, Niu L, et al. Purification, partial characteri-
zation, crystallization and structural determination of AHP-
LAAO, a novel l-amino-acid oxidase with cell apoptosis-
inducing activity from Agkistrodon halys pallas venom. Acta
Cryst D. 2004;60:974–977.
27. Georgieva D, Murakami M, Perband M, Arni R, Betzel C. The
structure of a native L-amino acid oxidase, the major compo-
nent of the Vipera ammodytes ammodytes venomic, reveals
dynamic active site and quaternary structure stabilization by
divalent ions. Mol Biosyst. 2011;7:379–384.
28. Arima J, Tamura T, Kusakabe H, et al. Recombinant expres-
sion, biochemical characterization and stabilization through
proteolysis of an L-glutamate oxidase from Streptomyces sp. X-
119-6. J Biochem. 2003;134:805–812.
29. Soda K. Microdetermination of D-amino acids and D-amino acid
oxidase activity with 3-methyl-2-benzothiazolone hydrazone
hydrochloride. Anal Biochem. 1968;25:228–235.
30. Battye TGG, Kontogiannis L, Johnson O, Powell HR,
Leslie AGW. iMOSFLM: A new graphical interface for diffraction
image processing with MOSFLM. Acta Cryst D. 2011;67:271–281.
31. Evans P. Scaling and assessment of data quality. Acta Cryst D.
2006;62:72–82.
32. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD,
Storoni LC, Read RJ. Phaser crystallographic software. J Appl
Cryst. 2007;40:658–674.
4. Böger RH, Bode-Böger SM. The clinical pharmacology of L-
Arginine. Annu Rev Pharmacol Toxicol. 2001;41:79–99.
5. Marescau B, Dedeyn PP, Lowenthal A, et al. Guanidino com-
pound analysis as a complementary diagnostic parameter for
hyperargininemia: Follow-up of guanidino compound levels
during therapy. Pediatric Res. 1990;27:297–303.
6. Vissers YL, Dejong CH, Luiking YC, Fearon KC, von
Meyenfeldt MF, Deutz NE. Plasma arginine concentrations are
reduced in cancer patients: Evidence for arginine deficiency?
Am J Clin Nutr. 2005;81:1142–1146.
7. Nikolelis DP, Hadjiioannou TP. Construction of an arginine
enzyme electrode and determination of arginine in biological
materials. Anal Chim Acta. 1983;147:33–39.
8. Liu D, Yin A, Ge K, Chen K, Nie L, Yao S. Enzymatic analysis
of arginine with the SAW/conductance sensor system. Enzyme
Microb Technol. 1995;17:856–863.
9. Tong SL, Rechnitz GA. Enzymatic L-arginine and L-lysine
determination using a carbonate ion selective membrane elec-
trode. Anal Lett. 1976;9:1–11.
10. Kameya M, Asano Y. Rapid enzymatic assays for L-citrulline
and L-argininebased on the platform of pyrophosphate detec-
tion. Enzyme Microb Technol. 2014;57:36–41.
11. Matsui D, Terai A, Asano Y. L-arginine oxidase from Pseudomo-
nas sp. TPU 7192: Characterization, gene cloning, heterologous
expression, and application to L-arginine determination.
Enzyme Microb Technol. 2016;82:151–157.
12. Pollegioni L, Motta P, Molla G. L-amino acid oxidase as biocatalyst:
A dream too far? Appl Microbiol Biotechnol. 2013;97:9323–9341.
13. Yu Z, Qiao H. Advances in non-snake venom L-amino acid oxi-
dase. Appl Biochem Biotechnol. 2012;167:1–13.
14. Kusakabe H, Midorikawa Y, Fujishima T, Kuninaka A,
Yoshino H. Purification and properties of a new enzyme, L-glu-
tamate oxidase, from Streptomyces sp. X-119-6 grown on wheat
bran. Agr Biol Chem. 1983;47:1323–1328.
15. Kusakabe H, Kodama K, Kuninaka A, Yoshino H, Misono H,
Soda K. A new antitumor enzyme, L-lysine oxidase from
Trichoderma viride. Purification and enzymological properties.
J Biol Chem. 1980;255:976–981.
16. Amano M, Mizuguchi H, Sano T, et al. Recombinant expres-
sion, molecular characterization and crystal structure of anti-
tumor enzyme, L-lysine α-oxidase from Trichoderma viride.
J Biochem. 2015;157:549–559.
17. Koyama H. Purification and characterization of a novel L-phe-
nylalanine oxidase (deaminating and decarboxylating) from
Pseudomonas sp. P-501. J Biochem. 1982;92:1235–1240.
18. Koyama H. Further characterization of a novel L-phenylalanine
oxidase (deaminating and decarboxylating) from Pseudomonas
sp. P-501. J Biochem. 1983;93:1313–1319.
33. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and
development of coot. Acta Cryst D. 2010;66:486–501.
34. Adams PD, Afonine PV, Bunkóczi G, et al. PHENIX: A com-
prehensive python-based system for macromolecular structure
solution. Acta Cryst D. 2010;58:1948–1954.
How to cite this article: Yano Y, Matsuo S, Ito N,
et al. A new L-arginine oxidase engineered from L-
glutamate oxidase. Protein Science. 2021;30:
1044–1055. https://doi.org/10.1002/pro.4070
19. Upadhyay S, Ohgami N, Kusakabe H, et al. Performance char-
acterization of recombinant L-glutamate oxidase in a micro
GOT/GPT sensing system. Sens Actuators B Chem. 2006;119:
570–576.