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 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 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, L-amino acid oxidase; LGOX, L-glutamate oxidase; PAO, polyamine oxidase. 3894 FEBS Journal 276 (2009) 3894–3903 ª 2009 The Authors Journal compilation ª 2009 FEBS Introduction l-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; 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 m = 5.0 mm). Artificial enzy- matic proteolysis of the precursor with a metallo- protease from Streptomyces griseus forms the mature enzyme with the a 2 b 2 c 2 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 a 2 b 2 c 2 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 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- 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 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 3895 mass 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 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 ˚ . 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 AB 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. 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 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|F o – F c | ⁄ 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) 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 ˚ ; angle, 1.5° No. of protein residues 597 of 628 Chemical components PO 4 , 4; FAD, 1 B-factor (A ˚ 2 ) All atoms 34.3 Protein atoms 34.3 Main chain atoms 33.87 Side chain atoms 34.78 FAD 21.85 PO 4 62.49 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-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 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. 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 3899 We 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. Crystal structure of L-glutamate oxidase J. Arima et al. 3900 FEBS Journal 276 (2009) 3894–3903 ª 2009 The Authors Journal compilation ª 2009 FEBS substrate specificity towards l-glutamate. This differ- 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 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 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 (a 2 b 2 c 2 ), 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 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. Experimental procedures Protein purification The LGOX precursor was purified from the cell lysate of E. coli JM109 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 60 °C 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 SDS ⁄ PAGE and MALDI-TOF MS (Autoflex II TOF ⁄ TOF; Bruker Daltonics Inc., Bremen, Germany). SDS ⁄ 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. 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 3901 Determination of N-terminal amino acid sequence The purified enzymes were blotted onto a poly(vinylidene 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- taining 1200 mm NaH 2 PO 4 , 800 mm K 2 HPO 4 , 200 mm LiSO 4 100 mm Caps (pH 10.5), and 20% glycerol, and flash-cooled in a nitrogen stream at 100 K. The LGOX crystals were of hexagonal space group P6 1 22, with the following unit cell dimensions: a = b = 123.88 A ˚ , c = 168.76 A ˚ . A dataset was collected at Spring-8 BL24 (Hyogo, Japan). Diffraction data were processed using the 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 (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. References 1 Beart PM (1975) An evaluation of l-glutamate as the transmitter released from optic nerve terminals of the pigeon. Brain Res 110, 99–114. 2 Collingridge GL & Lester AJ (1989) Excitatory amino acids receptors in the vertebrate central nervous system. Pharmacol Rev 40, 143–209. 3 Meldrum BS & Garthwate J (1990) Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Neurosci 11, 379–387. 4 Kusakabe H, Midorikawa T, Fujishima A, Kuninaka A & Yoshino H (1983) Purification and purification of a new enzyme, l-glutamate oxidase, from Streptomyces sp. X-119-6 grown on wheat bran. 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