The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an a2b2c2 structure; enzymatic proteolysis of the precursor forms the stable mature e
Trang 1Streptomyces sp X-119-6
Jiro Arima1,2,*, Chiduko Sasaki3,*, Chika Sakaguchi1, Hiroshi Mizuno1, Takashi Tamura1,
Akiko Kashima3, Hitoshi Kusakabe4, Shigetoshi Sugio3and Kenji Inagaki1
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
K Inagaki, Department of Biofunctional
Chemistry, Graduate School of Natural
Science and Technology, Okayama
University, Okayama-shi, Okayama
700-8530, Japan
Fax: +81 86 251 8299
Tel: +81 86 251 8299
E-mail: kinagaki@cc.okayama-u.ac.jp
S Sugio, Innovation Center Yokohama
Center, Mitsubishi Chemical
Corporation, Aoba-ku, Yokohama
227-8502, Japan
Fax: +81 45 963 4206
Tel: +81 45 963 3663
E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp
*These authors contributed equally to this
work
Database
Coordinate and structure factors of the
mature L -glutamate oxidase have been
deposited in the Protein Data Bank at
the Research Collaboratory for Structural
Bioinformatics under code 2E1M
(Received 26 February 2009, revised 30
April 2009, accepted 18 May 2009)
doi:10.1111/j.1742-4658.2009.07103.x
l-Glutamate oxidase (LGOX) from Streptomyces sp X-119-6, which cata-lyzes the oxidative deamination of l-glutamate, has attracted increasing attention as a component of amperometric l-glutamate sensors used in the food industry and clinical biochemistry The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an
a2b2c2 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 ) by x-ray crystallography ( MI:0114 )
Abbreviations
AB, o-aminobenzoate; LAAO, L -amino acid oxidase; LGOX, L -glutamate oxidase; PAO, polyamine oxidase.
Trang 2l-Glutamate has a flavor-enhancing activity that
cre-ates the sensation of ‘umami’; the monosodium salt of
l-glutamate is widely used as a seasoning for cooking
and as a food additive The amino acid is also the
principal excitatory neurotransmitter in the brain [1,2]
Furthermore, its excessive release might play a major
role in the neuronal death associated with various
neurological disorders [3] Therefore, the quantitative
assay of l-glutamate is important in the fields of food
production and clinical biochemistry
Actually, l-glutamate oxidase (LGOX; EC 1.4.3.11)
was first purified from an aqueous extract of a wheat
bran culture of Streptomyces sp X-119-6 [4] Along
with the production of ammonia and hydrogen
perox-ide via an imino acid intermediate, LGOX catalyzes
the oxidative deamination of the a-amino group of
l-glutamate to 2-ketoglutarate, A simple photometric
l-glutamate assay kit and amperometric l-glutamate
sensors using LGOX, based on colorimetric and
elec-trochemical measurements of hydrogen peroxide, are
commercially available The biosensor is now a useful
tool for in vitro and in vivo monitoring of mammalian
brain l-glutamate [5–7] In addition, results of recent
studies show that microfluidic biosensors using LGOX
are applicable for the detection of glutamic
oxaloace-tic transaminase, glutamic pyruvic transaminase and
c-glutamyl transpeptidase activities, which are
diagnos-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; kcat= 75 s)1; Km= 0.23 mm) A
peculiarity of LGOX from Streptomyces sp X-119-6 is
that the enzyme has a hexameric structure, a2b2c2; 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 (kcat= 33 s)1), and has low
affinity for substrate (Km= 5.0 mm) Artificial
enzy-matic proteolysis of the precursor with a
metallo-protease from Streptomyces griseus forms the mature
enzyme with the a2b2c2 structure without separation of
large proteolytic fragments The findings show that
LGOX is expressed in nature as the precursor in an
incompletely active form; the enzyme is digested by an
endopeptidase to yield the active form with an a2b2c2
oligomeric structure The mechanism of this structural
change, with its improvement in properties, is of
inter-est for basic enzymological studies
Another peculiarity of LGOX is its strict specificity for l-glutamate In addition to LGOX, several
l-amino acid oxidases (LAAOs) with strict specificity have been identified from various microorganisms:
l-lysine-a-oxidase [14], l-phenylalanine oxidase [15,16], and l-aspartate oxidase [17] These enzymes are considered to have an identical catalytic mechanism 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 a2b2c2 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-ments corresponding approximately to 42, 40, 17 and
10 kDa The N-terminal sequences of the 42 kDa and
40 kDa fragments were established to be ANEMT, indicating that both fragments were identified as an a-fragment The sum of the masses of the a-fragment, b-fragment and c-fragment of mature LGOX was approximately 70 kDa, indicating that short pro-teolytic fragments were separated from the LGOX molecule through digestion
On the basis of the molecular mass, we predicted the region of the respective fragments of mature LGOX The regions and the theoretical molecular masses of the respective fragments are presented in Fig 2A MS analysis of the smaller a-fragment showed a mass of approximately 39 900 Da (Fig 1B), indicating that the C-terminus of this fragment is located at approxi-mately residue 370 The larger fragment showed a
Trang 3mass of approximately 41 700 Da (Fig 1B), indicating
that the C-terminus lies closer to the N-terminus of the
c-fragment (Tyr390) We infer that the larger
a-frag-ment is an intermediate formed during protein
matura-tion An MS analysis of the c-fragment showed a
sharp peak with a molecular mass of 10 570 Da
(Fig 1B), thereby enabling identification of the
posi-tion of the C-terminal end of this fragment (Ala480)
The results show deletions of 40 residues between the
c-fragment and b-fragment and approximately 20
residues after the C-terminus of the b-fragment The
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 5C 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 Rfree of 24.8% and 30.8%, respectively, in the resolution range 45.3–2.8 A˚
Overall structure The overall structure is shown in Fig 3A The mature LGOX comprises an oligomeric dimer, with each pro-tomer containing three fragments (abc) and a bound FAD The protomer in the crystal asymmetric unit forms a biological dimer with its own symmetry equiv-alent, and interacts in a head-to-tail orientation with the substrate-binding site facing away from the dimer interface Within a single protomer, the chain of each fragment is substantially entangled with the other chains (Fig 3A) This finding is in agreement with the endopeptidase modification that occurs after protein folding The electron densities for amino acids 1–17, 364–376, 387–390, 481–522 and 674–701 are not visi-ble; crystallographic analysis revealed approximate cleavage sites on the single-polypeptide LGOX The FAD prosthetic group is buried deep within the enzyme It undergoes extensive interactions with the protein residues The FAD adopts a conformation resembling that seen in other FAD enzymes In the LGOX structure, the domain interacting with the dinu-cleotide moiety of FAD comprises six discontinuous regions of the structure: Val64–Gly70, Leu87–Ile98
Fig 1 SDS ⁄ PAGE and MS analysis of LGOX (A) SDS ⁄ PAGE of LGOX Lane M includes molecular weight protein markers Lanes 1 and 2, respectively, include precur-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.
Trang 4B
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 highhigh-lighted 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 #.
Trang 5and 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
domain
Structure analysis revealed two funnel-shaped
entrances (1 and 2 in Fig 4A) extending from the
surface of the protein into the interior, terminating at
the active site near to the FAD cofactor The presence
of the long funnel is a charcteristic of a number of
fla-voenzymes The funnel of LGOX is composed mainly
of the residues of the a-fragment The residues
com-posing the active site are contained in the a-fragment
and b-fragment The LGOX surface has two funnel
entrances (entrances 1 and 2 in Fig 4A) Both
entrances were observed to be approximately 20 A˚
from the active site
Structural comparison with LAAO
A 3D molecular structure comparison using the dali [20] program revealed that the overall structure of LGOX resembles those of LAAO with Z = 41.3
Table 1 Data collection and refinement statistics for the crystal
structure of mature LGOX R-merge = R(|(I – <I>)|) ⁄ R(I).
R = R|Fo– Fc| ⁄ R|F o |.
Crystal cell parameter
Unit cell parameter
a, b, c (A ˚ ) 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)
Rmerg(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 ˚ ; angle, 1.5
No of protein residues 597 of 628
Chemical components PO 4 , 4; FAD, 1
B-factor (A˚2)
Protein atoms 34.3
Main chain atoms 33.87
Side chain atoms 34.78
Ramachandran plot
Most favored (%) 85.6
Additional allowed (%) 12.8
Generously allowed (%) 1
Disallowed (%) 0.6
A
B
Fig 3 Overall structure and local structure around FAD of the mature LGOX (A) The functional hexamer with two protomers (a 2 b 2 c 2 ) On the left-hand side of the protomer, a-fragments, b-frag-ments and c-fragb-frag-ments 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 resiresi-dues
in a-fragments, b-fragments and c-fragments are, respectively, colored orange, green, and blue.
Trang 6(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 are presented in Fig 4B Through comparison of the enzymes’ funnel shapes, the cavity of the active site (space around the N5 of FAD) of LGOX was found
to be narrower than that of LAAO A structural study of PAO also revealed a U-shaped funnel, which
is more complicated than that of LAAO [21] The LGOX funnel shape and length resemble those of PAO (30 A˚) (Fig 4A) However, the comparison also revealed a narrower active site in LGOX than
in PAO
A
B
Fig 4 Structural comparisons of the funnels of LGOX, LAAO, and PAO, and the residues composing two entrances of the funnel of LGOX (A) Stereo view of funnels of LAAO, PAO, and LGOX The surfaces of the enzyme molecules are colored green, and the space that can accommodate a substrate in the funnel is shown as a gray tube This tube is funnel-shaped Black arrowheads indicate funnel entrances (B) Stereo view of the residues composing two entrances of the funnel of LGOX The residues associated with the construction of the entrances are presented as coils and sticks, colored according to the atom type FAD is shown in the CPK color scheme.
Trang 7We next examined the structural differences between
LGOX and LAAO The active site residues of LGOX
were superimposed on the model of LAAO containing
o-aminobenzoate (AB) as a ligand (Fig 6A) Several
residues composing the active sites of both enzymes
are mutually identical The residues corresponding to
Ile374 and Gly212 of LAAO are, respectively, Trp564
and Arg305 in LGOX Because of the bulkiness of the 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
Fig 6 Comparison of the active sites of LGOX, LAAO, and D -amino acid oxidase (A) Stereo view of the active site of LGOX superimposed on that of LAAO (Protein Data Bank code: 1F8S) (B) Stereo view of the active site of LGOX superimposed on that of D -amino acid oxidase (Protein Data Bank code: 1C8I) The regions of their active sites were superimposed along the isoallox-azine ring The residues of LGOX are shown
as blue sticks; those of LAAO and D -amino acid oxidase are shown as green sticks AB molecules observed in the structure of LAAO–AB and D -amino acid oxidase–AB complexes are shown as sticks colored according to the atom type FAD is shown
in the CPK color scheme The black arrow-head indicates N5 of the isoalloxazine ring.
Trang 8substrate specificity towards l-glutamate This
ence in specificity is probably associated with the
differ-ences in conformations of the active sites, as well as
entry points into the structures of the enzymes Pawelek
et al reported that, in the crystal structure of the
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 O2
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
assist hydride transfer and to be important for substrate
entry [18] Because there is no equivalent residue around
Gly316 of LGOX, further studies are necessary to
clar-ify which residue assists hydride transfer In addition to
the replacement of His223 by Gly, residue Glu219 of
LAAO is replaced by His312 in LGOX (Fig 6A)
Therefore, along with the architecture of the active site,
the residue substitutions between the two enzymes alter
the electrostatic environment significantly These
obser-vations suggest that the differences in amino acid
resi-dues engender differences in the electrostatic and steric
environments around the active sites of both enzymes
and result in differences in substrate specificity
In addition to the substrate specificity, LGOX
exhib-its a hexameric structure (a2b2c2), induced by
endo-peptidase digestions The LGOX precursor with a
homodimeric structure has a propensity to aggregate
Furthermore, it exhibits low thermal stability, low
catalytic activity, and poor substrate affinity Chen et al
reported that, by the recombinant expression of LGOX from Streptomyces platensis using Streptomyces lividans, the enzyme was expressed in S lividans cells as a precur-sor Moreover, the mature enzyme modified by endo-peptidase is observed in the extracellular fraction [12]
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 a2b2c2oligomeric structure after secretion The present study demonstrated that the artificial enzymatic proteolysis of the precursor forms the a2b2c2 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
Experimental procedures
Protein purification
The LGOX precursor was purified from the cell lysate of
E coliJM109 harboring the plasmid for LGOX production, pKK–LGOX, as described by Arima et al [13] The purified single-chain precursor was treated with the metalloprotease from S griseus (Sigma-Aldrich Corp., St Louis, MO, USA)
at room temperature for 4 h Then it was heated at 60C for
30 min to inactivate the protease After centrifugation (12 000 g, 10 min), the solution was loaded onto a column (DEAE-Toyopearl 650; Tosoh Corp., Tokyo, Japan) equili-brated with 20 mm potassium phosphate buffer (KPB) (pH 7.4) containing 0.1 m NaCl 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 purified mature enzyme sample
Molecular mass of LGOX
The molecular mass of LGOX was determined using
PAGE was performed with a 12% gel under denaturing conditions [26] For MS analysis, the enzyme (1 mg⁄ mL) was dialyzed against Milli-Q water The dialysate was then mixed with the MALDI matrix solution The mixture was then used for MS analysis
Trang 9Determination of N-terminal amino acid
sequence
The purified enzymes were blotted onto a poly(vinylidene
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 5C using the
sit-ting drop vapor diffusion method by mixing a protein
NaH2PO4, 800 mm K2HPO4, 200 mm LiSO4, 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
flash-cooled in a nitrogen stream at 100 K The LGOX
crystals were of hexagonal space group P6122, with the
c= 168.76 A˚
A dataset was collected at Spring-8 BL24 (Hyogo,
HKL2000 [27] The structure was solved using molecular
replacement with amore [28] with the structure of LAAO
from snake venom (Protein Data Bank code: 1F8S) The
resulting electron density maps were of sufficiently good
quality to trace the polypeptide chain
Crystallographic refinement
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 (a2b2c2) are mutually related by their
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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