Structural characterization of L-glutamate oxidase from
Streptomyces sp. X-119-6
Jiro Arima
1,2,
*, Chiduko Sasaki
3,
*, Chika Sakaguchi
1
, Hiroshi Mizuno
1
, Takashi Tamura
1
,
Akiko Kashima
3
, Hitoshi Kusakabe
4
, Shigetoshi Sugio
3
and Kenji Inagaki
1
1 Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Japan
2 Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Japan
3 Innovation Center Yokohama Center, Mitsubishi Chemical Corporation, Aoba-ku, Yokohama, Japan
4 Enzyme Sensor Co. Ltd, Liaison Center 311, University of Tsukuba, Japan
Keywords
L-amino acid oxidase; L-glutamate
oxidase; Streptomyces; substrate
specificity; X-ray crystallographic
structure
Correspondence

food industry and clinical biochemistry. The precursor of LGOX, which
has a homodimeric structure, is less active than the mature enzyme with an
a
2
b
2
c
2
structure; enzymatic proteolysis of the precursor forms the stable
mature enzyme. We solved the crystal structure of mature LGOX using
molecular replacement with a structurally homologous model of l-amino
acid oxidase (LAAO) from snake venom: LGOX has a deeply buried active
site and two entrances from the surface of the protein into the active site.
Comparison of the LGOX structure with that of LAAO revealed that
LGOX has three regions that are absent from the LAAO structure, one of
which is involved in the formation of the entrance. Furthermore, the
arrangement of the residues composing the active site differs between
LGOX and LAAO, and the active site of LGOX is narrower than that of
LAAO. Results of the comparative analyses described herein raise the
possibility that such a unique structure of LGOX is associated with its
substrate specificity.
Structured digital abstract
l
MINT-7041556: LGOX_gamma fragment (uniprotkb:Q8L3C7), LGOX_beta fragment
(uniprotkb:
Q8L3C7) and LGOX_alpha fragment (uniprotkb:Q8L3C7) physically interact
(
MI:0915)byx-ray crystallography (MI:0114)
Abbreviations
AB, o-aminobenzoate; LAAO,

tic markers of liver function [8,9].
LGOXs of several kinds have been identified from
the genus Streptomyces [4,10–12]. An enzyme from
Streptomyces sp. X-119-6 is the sole commercially
available enzyme that is useful for biosensors: it has
high substrate specificity and high stability (thermal
stability, 80 °C; k
cat
=75s
)1
; K
m
= 0.23 mm). A
peculiarity of LGOX from Streptomyces sp. X-119-6 is
that the enzyme has a hexameric structure, a
2
b
2
c
2
; the
precursor has been shown by recombinant expression
to have a homodimeric structure [13]. The precursor
tends to aggregate, has low thermal stability (40 °C),
has low catalytic activity (k
cat
=33s
)1
), and has low
affinity for substrate (K

but different affinities for amino acid substrates. In
this class of enzymes, the crystal structures of the
LAAO from snake venom and aspartate oxidase from
Escherichia coli are available [18,19]. However, little
information related to the determinant of substrate
specificity of the enzymes is available.
For this study, to investigate the relationship
between biochemical characteristics and structural fea-
tures of LGOX from Streptomyces sp. X-119-6, we
determined its molecular mass and analyzed its
detailed structure. Herein, we describe the crystal
structure of the mature enzyme and compare it with
the LAAO structure. On the basis of comparative
analyses, insights into the structural factors for the
biochemical characteristics of LGOX are discussed.
Results
Molecular mass of LGOX
Our previous study showed that LGOX is expressed as
a single polypeptide precursor in an incompletely
active form. It forms a mature enzyme with a hexa-
meric a
2
b
2
c
2
structure formed through protease modi-
fication [13]. As shown in Fig. 1, the LGOX precursor
has a molecular mass of approximately 75 kDa,
whereas the mature form of LGOX shows four frag-

difference in properties between the precursor and
mature LGOX may be attributed to distortions of the
single-chain structure with extra amino acid segments
as described above.
Structure determination and structural quality
We attempted to crystallize LGOX as both the pre-
cursor and mature forms to obtain detailed structural
information. Crystals of mature LGOX were grown
at 5 °C using the sitting drop vapor diffusion
method. The LGOX crystals were formed in the pres-
ence of a-ketoglutarate. Nevertheless, in crystallo-
graphic analyses, no electron density corresponding
to the ligand was observed at the active site pocket.
Crystals of the precursor also formed. However, the
crystal quality of the precursor was inadequate for
determination of the structure. On the other hand,
protein crystals were never grown, under any condi-
tions, from LGOX solutions in the presence of either
l-glutamate, l-aspartate, or l-aspargine. Therefore,
we performed structural analysis using only mature
LGOX crystals. The data collection and refinement
statistics for the crystal structure of the mature
LGOX are presented in Table 1. The refined model
contains 356 residues in the a-fragment, 151 residues
in the b-fragment, 90 residues in the c-fragment, an
FAD, and four phosphate anions with an R-factor
and R
free
of 24.8% and 30.8%, respectively, in the
resolution range 45.3–2.8 A

sor LGOX and mature LGOX. The N-terminal
amino acid sequences of the a-fragments,
b-fragments and c-fragments are shown
at the side of the panel. (B) MS analysis of
LGOX. Upper panel, LGOX precursor;
middle panel, a-fragment; lower panel,
b-fragment and c-fragment.
Crystal structure of
L-glutamate oxidase J. Arima et al.
3896 FEBS Journal 276 (2009) 3894–3903 ª 2009 The Authors Journal compilation ª 2009 FEBS
A
B
Fig. 2. Amino acid sequence of LGOX and structure-based sequence alignment of LGOX with LAAO. (A) The sequence is the primary struc-
ture deduced from the nucleotide sequence of LGOX. The region of the a-fragment is highlighted in black, that of the c-fragment is high-
lighted in dark gray, and that of the b-fragment is highlighted in light gray. Identified protease cleavage positions are indicated by black
arrowheads. The dotted line under the sequence shows a possible cleavage position, as identified using MS analysis. The theoretical values
of molecular masses of respective fragments are shown. The value presented in parenthese3s is the theoretical molecular mass of a smaller
fragment of the a-fragment. (B) Structure-based sequence alignment of LGOX with LAAO. The N-terminal residues of the a-fragments and b
fragments for which no electron density was observed are presented in lower-case letters. The secondary structural elements are indicated
by cylinders showing the a -helices and arrows indicating b-strands with numbering of the secondary structure. Residues conserved between
both enzymes are highlighted in black. Functionally similar residues are highlighted in gray. The residues composing the active site are indi-
cated by #.
J. Arima et al. Crystal structure of
L-glutamate oxidase
FEBS Journal 276 (2009) 3894–3903 ª 2009 The Authors Journal compilation ª 2009 FEBS 3897
and Gln352–Met354 in the a-fragment, Thr404–Ser409
in the c-fragment, and Tyr613–Gly616 and Gly644–
Glu645 in the b-fragment (Fig. 3B). The isoalloxazine
ring of FAD is positioned at the interface between the
FAD-binding domain and the substrate-binding

˚
) 123.88, 123.88, 168.76
a, b, c (°) 90, 90, 120
Space group P6122 (178)
Relative molecular mass 77 804
Collection and reduction
Wavelength (A
˚
) 1.0000
Resolution limit (A
˚
) 2.6
No. of total reflections 244 533
No. of unique reflections 24 388
Completeness (last shell) (%) 100 (100)
I ⁄ r 29.7
R
merg
(last shell) (%) 8.2 (51.4)
Refinement
Resolution range (A
˚
) 45.3–2.8
No. of unique reflections 19146
R (R
free
) (%) 24.8 (30.8)
rmsd (A
˚
) Bonds, 0.008 A

2
c
2
). On the left-hand side of the protomer, a-fragments, b-frag-
ments and c-fragments are, respectively, colored orange, green,
and blue. FAD is shown in the CPK color scheme. The N-terminals
of b-fragments and c-fragments and the C-terminals of a-fragments
and c-fragments are indicated by arrows. (B) View of LGOX in the
region of the FAD prosthetic group. The protein main chain is repre-
sented as a coil; FAD is shown as a stick. Side chains of the resi-
dues around FAD are depicted as wires. The regions and residues
in a -fragments, b-fragments and c-fragments are, respectively,
colored orange, green, and blue.
Crystal structure of
L-glutamate oxidase J. Arima et al.
3898 FEBS Journal 276 (2009) 3894–3903 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Fig. 5) and polyamine oxidase (PAO) with Z = 25.7,
although the primary structure of LGOX exhibits
approximately 20% and 12% identity, respectively,
with those of LAAO and PAO. The alignments of
sequences and secondary structure elements of LGOX
and LAAO are presented in Fig. 2B. Through struc-
tural comparison, we located three insertions in
LGOX, Asp150–Asn192, Ser246–Trp262, and Thr450–
Ala480, which were not found in the structure of
LAAO (Figs 2B and 5). These regions exist on the sur-
face of LGOX. In fact, the Asp150–Asn192 region is
involved in the formation of entrance 2 of the funnel.
The LGOX funnel shape is more complicated than
that of LAAO (Fig. 4A). Residues at both entrances

residues, the active site of LGOX is narrower than that
of LAAO. Moreover, the polar residues Glu219 and
His223 of LAAO are, respectively, replaced by His
and Gly in LGOX (His312 and Gly316 in Fig. 6A).
The differences described above partially explain their
different substrate specificities.
Discussion
This study revealed the crystal structure of mature
LGOX. The results show that the structure of LGOX
resembles that of LAAO. Structural comparison
revealed several differences between LGOX and LAAO:
LGOX has three regions on the surface that were not
found in the LAAO structure (Fig. 5); differences also
exist in funnel formation (Fig. 4A) and the arrangement
of the residues composing the active sites (Fig. 6A).
Comparison of the arrangement of the active site res-
idues of LGOX, LAAO and d-amino acid oxidase
revealed that the residues of LGOX are more similar to
those found in LAAO (Figs A and 6B), suggesting that
the arrangements of active site residues of LGOX and
LAAO are responsible for strict enantioselectivity. In
fact, LAAO can oxidize a wide range of hydrophobic
amino acids [22,23]. In contrast, LGOX exhibits strict
Fig. 5. Structural comparison of the overall structures of LGOX and
LAAO. The structure of LGOX is shown as an orange coil; regions
that cannot be found in the structure of LAAO are shown as blue
coils and sticks. The LAAO structure is shown as a light green coil.
FAD is shown in the CPK color scheme.
A
B

LAAO–AB complex, three AB molecules are visible
within the funnel [18]. On the basis of that observation,
they proposed the trajectory of the substrate to the
active site of LAAO. The structure of LAAO with its
substrate, l-phenylalanine, revealed a Y-shaped funnel
system [24]. It was suggested that the function of this
funnel was to allow the amino acid substrate and O
2
into the active site. In the LGOX structure, the two
funnel-shaped entrances lead from the surface to the
active site. The shapes of the LGOX and LAAO fun-
nels differ greatly (Fig. 4A). The LGOX funnel shape
resembles that of PAO (Fig. 4A). Previous reports of
the PAO structure show that its U-shaped funnel acts
as an entry and exit point for the substrate and product
[21]. Moreover, an exact match between the inhibitors
and the PAO funnel was revealed in the structure of
the PAO–inhibitor complex [25]. Similarly, we surmise
that the entrances of the funnel of LGOX have a dis-
tinctive function.
As portrayed in Fig. 6, the arrangements of many res-
idues composing the substrate-binding sites of both
LAAO and LGOX are similar. However, differences in
terms of the properties of their side chains are apparent
in several residues. The residues corresponding to Ile374
and Gly212 of LAAO are, respectively, Trp564 and
Arg305 in LGOX; consequently, the active site of
LGOX is narrower than that of LAAO. Moreover,
His223 of LAAO is replaced by Gly in LGOX (Gly316
in Fig. 6A). In fact, His223 of LAAO is expected to

We speculate that the LGOX activity that catalyzes the
oxidation of l-glutamate along with the production of
ammonia and hydrogen peroxide is toxic for or has a
negative influence on the growth of cells. Consequently,
it is considered that LGOX is present in cells as a pre-
cursor form that has low activity, and that the enzyme is
digested by an endopeptidase to yield the active form
with an a
2
b
2
c
2
oligomeric structure after secretion. The
present study demonstrated that the artificial enzymatic
proteolysis of the precursor forms the a
2
b
2
c
2
structure
without the separation of large proteolytic fragments.
Actually, the results of MS analysis indicate that the
LGOX precursor has a single-chain structure with two
extra regions (Fig. 2A). Further study of the structures
of LGOX, in addition to investigation of the precursor
form and LGOX–ligand complex, might shed light on
the detailed molecular characteristics associated with
the unique properties of LGOX.

difluoride) membrane after 12% SDS ⁄ PAGE under dena-
turing conditions. The membrane was then stained using
Coomassie brilliant blue. The protein band was excised
from the membrane. The protein bands were used to deter-
mine the N-terminal amino acid sequence through Edman
degradation.
Crystallization
Crystallization was performed using LGOX precursor and
mature forms. However, for the precursor form, crystals
sufficient for structure determination were not obtainable.
Crystals of mature LGOX were grown at 5 °C using the sit-
ting drop vapor diffusion method by mixing a protein solu-
tion [10 mg ⁄ mL protein in 20 mm KPB (pH 7.4) with
5mm dithiothreitol] and reservoir solution [1200 mm
NaH
2
PO
4
, 800 mm K
2
HPO
4
, 200 mm LiSO
4
, 100 mm Caps
(pH 6.2)] in a 1 : 2 ratio. Rod-shaped yellowish crystals
were grown in 3–4 weeks to sufficient size for use in diffrac-
tion studies.
Data collection and structure determination
The crystals were transferred into a harvest solution con-

Refinement of the structure was conducted using cnx [29]
against 2.8 A
˚
diffraction data. The final atomic model con-
tained a-fragments 18–363 and 377–386, c-fragment 391–
480, and b-fragment 523–673. The crystallographic R-factor
and free R-factor were 0.248 and 0.308, respectively
(Table 1). Analysis of crystal packing revealed that one
abc heterotrimer is involved in the asymmetric unit. Two
heterotrimers (a
2
b
2
c
2
) are mutually related by their crystal-
lographic two-fold symmetry representing the known
biological oligomerization state of LGOX with their own
symmetry equivalent.
Acknowledgements
This study was partly supported by a research grant
from the National Project on Protein Structural and
Functional Analysis from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan.
We thank T. Hatanaka, Research Institute for Biologi-
cal Sciences (RIBS), Okayama, for his kind help in
permitting us to use ICM software for structural
comparison.
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