Crystal structure of salt-tolerant glutaminase from Micrococcus luteus K-3 in the presence and absence of its product L-glutamate and its activator Tris Kazuaki Yoshimune1, Yasuo Shirakihara2, Mamoru Wakayama3 and Isao Yumoto1 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka, Japan Department of Biotechnology, Faculty of Life Science, Ritsumeikan University, Kusatsu, Shiga, Japan Keywords crystal structure; L-glutamate; Micrococcus luteus K-3; salt-tolerant glutaminase; Tris Correspondence K Yoshimune, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan Fax: +81 11 857 8980 Tel: +81 11 857 8444 E-mail: k.yoshimune@aist.go.jp Note Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 3ih8 (N), 3ih9 (T), 3iha (G), and 3ihb (TG) (Received September 2009, revised November 2009, accepted 26 November 2009) Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme Our previous study determined the structure of its major 42-kDa fragment Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product l-glutamate and its activator Tris, which activates the enzyme by sixfold With the exception of a ‘lid’ part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for l-glutamate (G), aa for Tris (T) and aa for both l-glutamate and Tris (TG) l-Glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure Comparison of the glutamate-binding site between Mglu and saltlabile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu A comparison of the substrate-binding pocket of Mglu, which is highly specific for l-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than l-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of l-asparagine with proper interactions Structured digital abstract l MINT-7305730: Mglu (uniprotkb:Q4U1A6) and Mglu (uniprotkb:Q4U1A6) bind (MI:0407) by x-ray crystallography (MI:0114) doi:10.1111/j.1742-4658.2009.07523.x Introduction Glutaminase (EC 3.5.1.2) hydrolyzes l-glutamine to produce l-glutamate and ammonia It is highly specific for l-glutamine and is distinct from asparaginase (EC 3.5.1.1), which hydrolyzes both glutamine and asparagine [1] Glutaminase is found in many organisms, including mammals, fungi, yeasts and bacteria [1–5] Glutaminase from Micrococcus luteus K-3 (Mglu) and AoGlS glutaminase from Aspergillus oryzae RIB40 are salt-tolerant [6,7] Glutaminase (YbaS) from Escherichia coli exhibits low b-lactamase activity [5], and Abbreviations aa, amino acid residues; F, major fragment of Mglu; G, presence of L-glutamate; Mglu, Micrococcus glutaminase; PDB, protein data bank; N, absence of additives; T, presence of Tris; TG, presence of Tris + L-glutamate 738 FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS K Yoshimune et al glutaminases from E coli [5,8], Bacillus subtilis [5], pigs [9] and humans [2] exhibit allosteric behavior for l-glutamine with positive cooperativity [2,8,9] Moreover, the crystal structures of a major fragment of Mglu [protein data bank (PDB) 3if5] [10], and glutaminases from E coli (PDB 1u60) [5], B subtilis (PDB 1mki, 3brm, 2osu) [5], Geobacillus kaustophilus (PDB 2pby) and human kidney (PDB 3czd) have been determined The structure of glutaminase from B subtilis (YbgJ, Bacillus glutaminase) with covalently bound 5-oxol-norleucine (PDB 3brm) reveals its Ser74 to be the catalytic nucleophile [5] On the basis of their crystal structures and the conserved amino acid residues (aa) of their active sites, these glutaminases are classified into a large group of bacterial penicillin-binding proteins and b-lactamases [5,10] Most enzymes from nonhalophilic microorganisms are inhibited in high salt concentrations [1,11] Mglu is a salt-tolerant enzyme with about 40% residual activity, even in 2.7 m NaCl [6,12] Salt-tolerant enzymes are distinct from halophilic enzymes that are inactive in the absence of salt [13] Although the structures of the fragments of Mglu [10] and algal carbonic anhydrase [14] have been determined, the mechanism of salt tolerance is not very well understood Mglu is composed of N-terminal (1-305 aa) and C-terminal (315456 aa) domains, with a peptide linker (306-314 aa) We previously reported the structure of a major fragment (referred to as F) of Mglu The fragment (F) consists of the conserved N-terminal domain that contains active-site residues and part of the C-terminal domain The structure of the N-terminal domain is conserved among species (E coli, B subtilis, G kaustophilus and human kidney) with superposition rmsd val˚ ues ranging from 1.2 to 1.5 A (when superimposed onto Mglu) The C-terminal domain is not present in other glutaminases, such as those from B subtilis and E coli [5,10]; therefore, it was hypothesized that the C-terminal domain might be responsible for salt tolerance [10] Here, we report the crystal structure of the intact glutaminase under four different conditions: in the absence of the additives (referred to as N); in the presence of Tris (referred to as T); in the presence of l-glutamate (referred to as G); and in the presence of both Tris and l-glutamate (referred to as TG) Furthermore, the liganded (TG) and unliganded (T) structures of Mglu were compared with those of salt-labile Bacillus glutaminase to gain insight into the mechanism of salt tolerance The liganded (TG) structure was also compared with that of Erwinia carotovora asparaginase, which shows a broader substrate specificity, to investigate the mechanism of the narrow substrate specificity Crystal structure of salt-tolerant glutaminase Results and Discussion Determination of overall structure In a previous study [10], we determined the F structure containing the truncated region of the C-terminal domain; however, we failed to determine the structure of the intact glutaminase because the crystals exhibited heavy twinning In the present study, we found new crystallization conditions that employed poly(ethylene glycol) 4000 as a precipitant (previously sodium citrate was used), and these crystallization conditions led us to determine nearly the full-length structure of Mglu in the presence and absence of l-glutamate and Tris (N, T, G and TG) l-Glutamate was identified in the G and TG structures, and Tris was identified only in the TG structure The crystallographic statistics are summarized in Table There were two molecules in the asymmetric unit of space group C2, and a physiological dimer [10] was generated by a twofold axis of crystallographic symmetry The two independent physiological dimers shared a similar structure The physiological dimer of the TG structure is shown in Fig The monomer structures were very similar among the four different forms However, small, but significant, differences were found to exist among those structures and these will be described in the subsequent sections Figure shows the C-terminal domain in the F, N, G, T and TG structures to demonstrate the structural similarity and varying degree of disordered regions that are present The N structure was found to have the largest number of disordered residues (354-376, 395-401 and 446-456 aa; 41 aa in total) Bound l-glutamate reduced the number of disordered residues (353-359, 397-402 and 449456 aa; 21 aa in total), and Tris dramatically reduced this number even further (449-456 aa; eight aa in total) In the presence of both Tris and l-glutamate, the number of disordered residues was minimal (451456 aa; six aa in total) A search for structural homologues of the C-terminal domain using the DALI [15] databases identified SpoIIAA (anti-anti-sigma factor; PDB 1til, chain D; 14% sequence identity) No structural homologue for any part (354-403 aa) of the C-terminal domain was found by the search SpoIIAA [16] (6-37 and 4381 aa) could be superimposed on parts of the C-terminal domain (319-350 and 404-442 aa) with rmsd ˚ values of 1.39 A SpoIIAA was found to bind to SpoIIAB, thereby antagonizing the anti-rF function of SpoIIAB; the structure of SpoIIAA, in complex with SpoIIAB, contained 11 interface residues (21, 23-25, FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS 739 Crystal structure of salt-tolerant glutaminase K Yoshimune et al Table Crystallographic statistics and refinement parameters X-ray source Data collection ˚ Resolution (A) Mean I ⁄ r (I)a No of reflections Measure Unique Completeness (%)a Rmerge (%)a,b Overall Highest-resolution shell Lowest-resolution shell Multiplicitya Unit-cell constants ˚ a (A) ˚ b (A) ˚ c (A) a (°) b (°) c (°) ˚ B value from Wilson plot (A2) Molecular replacement and refinement Initial model ˚ Resolution range (A) No of reflections R factor for 95% of datac Free R factor for 5% of data No of atoms Protein Water Glutamate Tris Disordered amino acid residues rmsd from ideality ˚ Bond length (A) Bond angle (°) ˚ Average B value (A2) Ramachandran analysis Favoured (%) Allowed (%) Generally allowed (%) Disallowed (%) ˚ Error in coordinate by Luzzati (A) N BL-6A T AR-NW12A G AR-NW12A TG BL-6A 2.3 10.9 (2.2) 2.5 11.0 (2.1) 2.6 9.1 (2.8) 2.4 8.7 (1.8) 189 811 52 671 100 (99.9) 149 207 40 451 98.4 (98.0) 125 111 35 920 98.2 (99.6) 147 220 46 819 99.5 (98.6) 11.1 54.6 6.1 3.6 (3.6) 12.4 49.2 8.2 3.7 (3.7) 11.9 35.6 8.7 3.5 (3.5) 13.1 41.2 5.7 3.1 (2.8) 118.08 142.25 74.25 90 104.1 90 42.1 117.70 141.39 75.23 90 104.5 90 52.8 117.45 141.62 75.15 90 104.0 90 58.0 118.36 141.20 76.12 90 105.4 90 41.0 F 19.9–2.3 52 579 0.247 0.291 TG 19.9–2.5 40 379 0.217 0.258 N 19.9–2.6 35 873 0.271 0.309 N 19.9–2.4 46 741 0.222 0.268 118 433 0 86 664 288 0 16 505 191 20 39 679 511 20 16 14 0.006 1.30 40.8 0.008 1.38 48.9 0.008 1.43 70.4 0.007 1.37 40.9 89.1 10.2 0.7 0.32 88.3 10.8 0.9 0.36 85.7 13.2 1.1 0.51 86.2 12.8 0.9 0.34 a Numbers in parentheses refer to data for the highest-resolution shell Rmerge = RhRi|I(h)-I(h)i| ⁄ RhRiI(h)I, where h is a unique reflection index, I(h)I is the intensity of symmetry-related reflections and I(h) is the mean intensity c R factor=Rh||Fo|h| ⁄ Rh|Fo|h, where h is a unique reflection index b 31, 58, 59, 61, 67, 89 and 90 aa) The structurally homologous residues in Mglu (334, 336-338, 344, 419, 420, 422 and 428 aa; nine aa in total) were located near the interface of the physiological dimer The Cterminal domain may contain structural features involved in protein–protein interactions to form the physiological dimers 740 Active site Figure shows l-glutamate in the G and TG structures The electron density of l-glutamate became more pronounced upon the addition of Tris The location and orientation of l-glutamate were determined on the basis of the lower electron density of FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS K Yoshimune et al Crystal structure of salt-tolerant glutaminase Fig The TG structure in the physiological dimeric form One monomer is shown in cyan and the other is shown in yellow L-Glutamate (magenta) and Tris (red) are shown as cylinders The ‘lid’ (26-29 aa) is shown in magenta Fig C-terminal domains in the F, N, G, T and TG structures The backbone atoms of the C-terminal domain (1-305 aa) in the F (black), G (yellow), T (cyan) and TG (magenta) structures are superimposed on that in the N structure (blue) The newly determined part of the C-terminal domain in the T and TG structures suggests a revised F structure (PDB 3if5) There are disordered regions in the F (354-403 and 449456 aa), N (354-376, 395-401 and 446-456 aa), G (353-359, 397-402 and 449-456 aa), T (449-456 aa) and TG (451-456 aa) structures Fig Active site of Mglu L-Glutamate and active-site residues in the G (yellow) and TG (magenta) structures are represented as cylinders Fo-Fc electron densities in the maps of the G (yellow) and TG (magenta) structures are contoured at +2.0 and +3.0 sigma levels, respectively Putative interactions ˚ (atom distance less than 3.9 A) are shown by dotted lines the a-amino group, and on the higher electron density of the a-carboxyl group, of l-glutamate Figure shows the seven residues (Gln63, Ser64, Asn114, Glu160, Asn167, Cys195 and Val261) that interact with l-glutamate, and also shows Lys67, which is ˚ probably hydrogen bonded (2.9 A) to Ser64 These FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS 741 Crystal structure of salt-tolerant glutaminase K Yoshimune et al residues are conserved among glutaminases from B subtilis (YbgJ) [5], E coli (YbaS) [5], G kaustophilus (PDB 2pby) and humans (PDB 3czd), except for Gln63, for which a glutamate residue is substituted in YbaS The C-a locations and side-chain orientations of the seven conserved residues are similar among these glutaminases, suggesting that these residues have similar functions On the basis of the proposed catalytic mechanism of Bacillus glutaminase, Ser64 is likely to be the putative catalytic nucleophile, and Lys67 may function as general base to assist Ser64 Tyr27 (Tyr37 in YbgJ) is hypothesized to interact with the a-amino group of l-glutamine, and Tyr191 (Tyr201 in YbgJ) and Tyr243 (Tyr253 in YbgJ) are proposed to participate in the proton-transfer reaction during the catalysis In addition to the seven conserved residues, these residues surround l-glutamate in the TG structure and they are also structurally conserved among these glutaminases Effect of L-glutamate on the structure Figure 4A shows the effect of l-glutamate on the structure of Mglu A comparison between the T and TG structures shows clearly that a single region (26-29 aa) changes upon l-glutamate binding We refer to this segment as the ‘lid’, which is located at the surface of gluta- minase and near its active site (Fig 1), because it appears to enclose the bound l-glutamate in the G and TG forms In the unliganded form, the lid is placed further from the active site, opening up the active site This open–close motion, caused by the presence of l-glutamate, is small, as shown in Fig 5; however, it is significant, as judged from the plot shown in Fig 4A The N and G structures were also compared to evaluate the l-glutamate effect Figure 4A shows the peak caused by the lid motion, described above, and two additional peaks (100-105, 303-309 aa) However, those two peaks were considered to be insignificant as they also appear in the N ⁄ T comparison shown in Fig 4B It seems that the N structure is structurally unique among all the structures at 100-105 aa and 303-309 aa This is supported by the fact that these two peaks are not observed in the F ⁄ G or F ⁄ T comparisons, in which the F form has no bound ligand (data not shown) It was difficult to determine what brought about the different conformations of the regions at 100-105 and 303-309 aa in the N form, and we investigated the following points First, these areas are not involved in any contacts with other molecules in crystals in all the crystal forms Second, the overall contact ˚ area of the N structure (3 890 A2) is significantly smal˚ ˚ ler than those of the T (4 583 A2), G (4 435 A2) and ˚ 2) structures This is apparently because TG (4 559 A Fig Effect of L-glutamate and Tris on backbone atoms Backbone atoms of the overall amino acid residues of the T and TG (T ⁄ TG), N and G (N ⁄ G), G and TG (G ⁄ TG), and N and T (N ⁄ T) structures are superimposed Effects of (A) L-glutamate (T ⁄ TG and N ⁄ G) and (B) Tris (G ⁄ TG and N ⁄ T) on the structure are shown Circles and numbers indicate residues mentioned in the text 742 FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS K Yoshimune et al Crystal structure of salt-tolerant glutaminase Fig Active sites of Mglu and Bacillus glutaminase in the presence and absence of ligands Backbone atoms of the seven conserved amino acid residues and the three conserved tyrosine residues in Mglu and Bacillus glutaminase are superimposed T (cyan) and TG (magenta) structures, and structures of Bacillus glutaminase in the presence (BD, grey) and absence (B, white) of 5-oxo-L-norleucine are ˚ shown in different colors Atoms of ligands are represented by a ball-and-stick model Putative interactions (atom distance less than 3.9 A) are shown by dotted lines Residues in the T structure (residue number and type) and in the BD structure (residue number) are labeled Residues 36-39 in the B structure (homologous residue of 26-29 in Mglu) are disordered of the more extensive disordered regions in the C-terminal domain of the N structure Salt-tolerant mechanism A comparison between the structures of Mglu and salt-labile glutaminase was expected to elucidate the structural features of salt-tolerant enzymes Bacillus glutaminase is a structural homologue of Mglu, and thus the enzyme is an appropriate counterpart As the salt tolerance of Bacillus glutaminase had not yet been determined, the gene was cloned and the overproduced enzyme was purified The purified Bacillus glutaminase exhibited no activity in 1.3 m NaCl at its optimum pH 8.0 (data not shown) Figure shows the active sites of Mglu and Bacillus glutaminase in the presence and absence of the ligands The l-glutamate of Mglu is located near the seven conserved amino acid residues (shown in Fig 3), the lid part (26-29 aa) and the three conserved tyrosine residues (Tyr27, Tyr191, and Tyr243) Homologous residues of the three conserved tyrosine residues (Tyr37, Tyr201, and Tyr253) in Bacillus glutaminase have been hypothesized to be important for the catalytic activity of the enzyme [5] The location of l-glutamate in the TG structure is similar to that of 5-oxo-l-norleucine, which covalently binds to Bacillus glutaminase Table shows the displacement, upon ligand binding, of the amino acid residues that are shown in Fig The displacement of these residues in Mglu is much smaller than those in Bacillus glutaminase In addition to the conserved amino acid residues, the displacement of the overall amino acid residues in Mglu is also small compared with those of Bacillus glutaminase Figure shows a comparison of Table Displacement of the backbone atoms of Mglu and Bacillus glutaminase induced by ligand binding Displacement values for backbone atoms of the nonliganded and liganded structures are shown The average and SD of these conserved residues (nine aa in total) are 0.16 ± 0.07 (Mglu) and 0.51 ± 0.34 (Bacillus glutaminase) Mglua Residue ˚ rmsd (A) Bacillus glutaminaseb ˚ Residue rmsd (A) The seven conserved residues that interact with L-glutamate Gln63 0.10 Gln73 0.30 Ser64 0.15 Ser74 0.29 Asn114 0.14 Asn126 0.57 Glu160 0.11 Glu170 0.57 Asn167 0.14 Asn177 0.94 Cys195 0.14 Cys205 0.30 Val261 0.17 Val271 0.25 Average ± SD 0.14 ± 0.02 0.46 ± 0.25 The three conserved tyrosine residues Tyr27c Tyr191 Tyr243 Average ± SD 1.43 0.16 0.33 0.25 ± 0.12 Tyr37 Tyr201 Tyr253 -d 0.20 1.16 0.68 ± 0.68 a Backbone atoms of the N-terminal domains (1-305 aa) in the T and TG structures are aligned b Backbone atoms of Bacillus glutaminase are superimposed on those of the liganded enzyme c The value of Tyr27 is excluded from the calculation of the average and SD d Tyr37 is disordered in the nonliganded structure of Bacillus glutaminase the displacement of all the backbone atoms of Mglu and Bacillus glutaminase induced by ligand binding Mglu shows no significant conformational change ˚ ˚ (average, 0.25 A; SD, 0.15 A) except for the lid motion ˚ ˚ of residues 26-29 (average, 1.14 A; SD, 0.38 A), which FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS 743 Crystal structure of salt-tolerant glutaminase K Yoshimune et al asparaginase in complex with l-glutamate (PDB 2hln) [18] Mglu binds l-glutamate in an extended form ˚ (Fig 7A) with a contact area of 288 A2 Asparaginase binds l-glutamate in a folded form (Fig 7B) in an apparently much smaller pocket with a contact area of ˚ 233 A2 These findings suggest that the substrate-binding pocket of Mglu is too large to bind l-asparagine with proper interactions Effect of Tris on the structure Fig Displacement of backbone atoms for all amino acid residues of Mglu and Bacillus glutaminases induced by ligand binding Displacements of the conserved amino acid residues in Table are indicated by circles (A) Backbone atoms of all amino acid residues in the T structure are superimposed on those in the TG structure The arrow indicates the Tyr27 residue The mean and SD of the val˚ ˚ ues are 0.25 A and 0.15 A, respectively (B) The backbone atoms of liganded and unliganded structures of Bacillus glutaminase are superimposed Displacement of 55 aa for Bacillus glutaminase (327 aa) is not shown because of the disordered regions in the ˚ ˚ structures The mean and SD of the values are 0.42 A and 0.37 A, respectively is described above By contrast, Bacillus glutaminase ˚ shows relatively large displacement (average, 0.42 A; ˚ ) The small conformational change is a SD, 0.37 A possible explanation for the salt-tolerant glutaminase As inactivation of nonhalophilic enzymes by high salt concentrations is usually caused by a decrease in flexibility [17], an enzyme reaction with a small conformational change might be favourable under high salt conditions However, this concept may be specific to glutaminase because such a small motion (average ˚ ˚ 0.24 A; SD 0.11 A) as a result of ligand binding is also observed in b-lactamase Toho-1 (PDB 1bza and 1iyq) from E coli Substrate specificity Glutaminase is strictly specific to l-glutamine [1] and distinct from asparaginase whose substrates are both glutamine and asparagine [18–23] To gain structural insights regarding substrate specificity, the TG structure was compared with the structure of E carotovora 744 Tris was found to activate Mglu by approximately sixfold at pH 7.5 (data not shown) This activation is specific for Tris, and Tris analogues, such as Bistris or tricine, induce no such effect Similar specific activations have also been observed in other proteins [24– 30] As shown in Fig 8, Tris is identified only in the TG structure Residues Arg223 and Ser227 interact with Tris Tris binding in the TG structure seems to be coupled to the protein local structure in the region encompassing aa 222-228, which is distinct from the corresponding structures in the N, T and G forms, in which Tris is not identified In contrast to l-glutamate, Tris binding induces a lesser degree of structural changes, as shown in the G ⁄ TG superposition plot in Fig 4B The N ⁄ T superposition is not taken into account for the reasons described in the previous section The N-terminal residues (Met1-His3)that give a single peak in the G ⁄ TG plot interact with the C-terminal domain residues (Asp345, Leu347, Thr352, Gly428 and Asp435) in the physiological dimer in all the structures Therefore, the observed structural difference in the N-terminal residues must have arisen from subtle differences in the interactions across the domain interface All the residues involved here seem to be unrelated to the Trisbinding site residues (including Arg223 and Ser227), either directly or indirectly Similarly, the 33 ordered residues in the T structure (that are disordered in the N form) are not related to the Tris-binding site residues in any way Thus, the effect of Tris binding on the glutaminase structure remains unclear Catalytic mechanism of Mglu The similar spatial arrangement of the probable activesite residues in Mglu and Bacillus glutaminase suggests that the catalytic mechanism of Mglu is similar to the proposed catalytic mechanism of Bacillus glutaminase [5] The structure of Bacillus glutaminase that covalently binds 5-oxo-l-norleucine (PDB 3brm) mimics that of the acyl-enzyme intermediate, while the Mglu TG structure may represent a stage just prior to prod- FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS K Yoshimune et al Crystal structure of salt-tolerant glutaminase A B Fig L-Glutamate in complex with Mglu and asparaginase of Erwinia carotovora LGlutamate (cylinder) on the surfaces of the structures of TG (A) and asparaginase of E carotovora (B) are shown Putative inter˚ actions (atom distance less than 3.9 A) are shown by dotted lines Fig Tris in the TG structure The Fo-Fc electron density in the map of the TG structure is contoured at the +2.4 sigma level Putative ˚ interactions (atom distance less than 3.9 A) are shown by dotted lines The backbone atoms of all amino acid residues in the N (blue), G (yellow) and T (cyan) structures are superimposed on those in the TG (magenta) structure uct release The acyl-enzyme intermediate may be formed by the nucleophilic attack of Ser64 (Ser74 in YbgJ) on the glutamine, which is assisted by the general base Lys67 (Lys77 in YbgJ) and Tyr243 (Tyr253 in YbgJ) as a mediator of proton transfer Deacylation may occur via nucleophilic attack by water, which is assisted by the general base Tyr191 (Tyr201 in YbgJ) In this study, the lid part containing Tyr27 (Tyr37 in YbgJ) was present in both ligand-free and l-glutamate-bound states The positions of the lid were distinct from that in the acyl-enzyme intermediate state (disordered in the ligand-free state in YbgJ) In the absence of the ligand, the lid part is separated from the active site, which leads to a more open active site Upon the binding of ligand in the acyl-enzyme intermediate state, the lid part is located very close to the active site, allowing interaction with the covalently bound 5-oxo-l-norleucine Before the release of l-glutamate, the lid may be in the intermediate position found in the Mglu TG structure By contrast, b-lactamase has no amino acid residue equivalent to the lid part, and shows no significant conformational change upon the binding of ligand [31] The smaller size of l-glutamine than the substrates for b-lactamase could be one of the reasons for the conformational changes of the lid part Experimental procedures Enzyme assay Glutaminase activity was assayed by determining the formation of l-glutamate by l-glutamate dehydrogenase, as previously described [12] The activity was assayed with 30 mm l-glutamine and 50 mm potassium phosphate buffer (pH 7.5) at 30 °C for 10 min, unless stated otherwise The pH values of Tris and l-glutamate were adjusted to the pH of the reaction mixture before addition to the mixture One FEBS Journal 277 (2010) 738–748 ª 2009 The Authors Journal compilation ª 2009 FEBS 745 Crystal structure of salt-tolerant glutaminase K Yoshimune et al unit of glutaminase was defined as the amount of enzyme that catalyzed the formation of lmol of l-glutamate per minute The protein concentration of the cell solution was estimated using the bicinchoninic acid (BCA) Protein Assay Reagent kit (Pierce Biotechnology, Rockford, IL, USA) with BSA as the standard Structural determination The recombinant Mglu was expressed and purified as described previously [32] The crystals of Mglu (N) were grown at 20 °C using the hanging-drop vapor-diffusion method with 0.75 mL of reservoir solution [90 mm HEPES buffer (pH 7.5), 180 mm sodium acetate and 27% (w ⁄ v) poly(ethylene glycol) 4000] and lL of protein solution [10 mgỈmL)1 of protein, 50 mm HEPES buffer (pH 7.5), 100 mm sodium acetate and 15% (w ⁄ v) poly(ethylene glycol) 4000] The protein solutions that additionally contained 0.3 m Tris (T), 0.2 m l-glutamate (G), and 0.3 m Tris + 0.2 m l-glutamate (TG) were prepared at pH values of 7.0, 7.5 and 7.2, respectively After treatment with paratone, the crystals were flash-frozen using liquid nitrogen Diffraction data were collected at beamlines at the Photon Factory, Tsukuba, Japan All data sets were collected at 95 K All images were indexed and integrated using the program MOSFLM [33], and the data sets were phased with molecular replacement using the program AMoRe [34] in the CCP4 program package [35] The F structure (PDB 3if5) [10] was used as an initial phasing model for the N structure The G and TG structures were solved using the N structure as a search model, and the TG structure was used in phasing of the T structure The structures were refined using the program CNS [36] with manual rebuilding using the program O [37] In CNS, the structures were refined using a combination of rigid-body, conjugate gradient minimization refinement and B-factor refinement The two molecules in the asymmetric units were refined without noncrystallographic symmetry restraints, which typically increased the free R values by 0.1% Although the R and free R values of the four structures were in the range of the values of the published structures, those of the N and G structures were slightly poor [38,39] For the N structure, the extensive disordered regions in its structure might be responsible for these values For the G structure, the relatively poor quality of the diffraction data sets was indicated by the slightly worse statistics of the data sets compared with the other structures, particularly Rmerge in the lowestresolution shell and the high B values from a Wilson plot (Table 1) In spite of these unfavourable values, the main chain and the side chain were clearly identified in the 2FoFc electron density map, and the final difference Fourier maps did not contain any significant peaks To confirm the validity of the structures, each of the four structures was fitted to each of the four diffraction data sets using rigidbody refinement All the four structures had minimum 746 R and free R values (each difference was in the range of 1–5%), when the structure was fitted to the cognate diffraction data sets The programs PROCHECK [40] and SFCHECK [41] in the CCP4 package were used for stereochemistry analysis of all models and for calculating the rmsd as well as the average error by the Luzzati plot The structures were superimposed on each other using the program SUPERPOSE [42] in the CCP4 package All the figures illustrating these structures were prepared using the program CCP4mg [43,44] The contact areas were calculated using AREAIMOL [45] in the CCP4 package ˚ with the use of the 1.4 A probe The coordinates have been deposited in the PDB under accession codes 3ih8 (N), 3ih9 (T), 3iha (G) and 3ihb (TG) Gene cloning and purification of Bacillus glutaminase The Bacillus glutaminase gene was amplified using the chromosomal DNA of B subtilis 168 (ATCC23857) The PCR product was digested with NdeI and SalI and ligated into the corresponding sites of pET21a (Novagen, San Diego, CA, USA) The Bacillus glutaminase gene in the resulting plasmid pYbgJ was confirmed to have the correct sequence The E coli BL21 (DE3) strain was transformed with pYbgJ, cultured at 37 °C for 12 h in Luria–Bertani (LB) medium supplemented with ampicillin (50 lgỈmL)1) and mm isopropyl thio-b-d-galactoside, and then sonicated in 10 mm Tris ⁄ HCl (pH 7.5) Bacillus glutaminase was purified using Q-sepharose (Tosoh, Tokyo, Japan) and hydroxyapatite (Wako, Osaka, Japan) The homogeneity of the final preparation of Bacillus glutaminase was confirmed by SDS ⁄ PAGE Acknowledgements Part of this study was supported by The Salt Science Research Foundation (no 0720) and the NIG Cooperative Research Program (A50) References Nandakumar R, Yoshimune K, Wakayama M & 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