The structure of hyperthermophilic β-N-acetylglucosaminidase reveals a novel dimer architecture associated with the active site.

The structure of hyperthermophilic b-Nacetylglucosaminidase reveals a novel dimer architecture associated with the active site Shouhei Mine1, Yuji Kado2,3, Masahiro Watanabe4, Yohta Fukuda3, Yoshito Abe5, Tadashi Ueda5, Yutaka Kawarabayasi1,6, Tsuyoshi Inoue3 and Kazuhiko Ishikawa4 1 2 3 4 5 6

National Institute of Advanced Industrial Science and Technology (AIST), Hyogo, Japan Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, Japan Graduate School of Engineering, Osaka University, Japan Biomass Refinery Research Center, AIST, Hiroshima, Japan Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Faculty of Agriculture, Kyushu University, Fukuoka, Japan

Keywords chitin; crystal structure; Nacetylglucosamine; thermostable enzyme; b-N-acetylglucosaminidase Correspondence S. Mine, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan Fax: +81 6 6491 5028 Tel: +81 72 751 9549 E-mail: [email protected] K. Ishikawa, Biomass Refinery Research Center, AIST, 3-11-32 Kagamiyama, HigashiHiroshima, Hiroshima 739-0046, Japan Fax: +81 72 751 9621 Tel: +81 82 493 6839 E-mail: [email protected] (Received 26 June 2014, revised 2 September 2014, accepted 11 September 2014) doi:10.1111/febs.13049

The b-N-acetylglucosaminidase from the hyperthermophilic bacteria Thermotoga maritima (NagA) hydrolyzes chitooligomers into monomer b-N-acetylglucosamine. Although NagA contains a highly conserved sequence motif found in glycoside hydrolase (GH) family 3, it can be distinguished from other GH family 3 b-N-acetylglucosaminidases by its substrate specificity and biological assembly. To investigate its unique structure around the active site, we The NagA determined the crystal structure of NagA at a resolution of 2.43 A. forms a dimer structure in which the monomer structure consists of an N- and a C-terminal domain. The dimer structure exhibits high solvation free energy for dimer formation. From mutagenesis analyses, the catalytic nucleophile and general acid–base residues were supposed to be Asp245 and His173, respectively. The most striking characteristic of NagA was that it forms the active site cleft from the N-terminal domain and the C-terminal domain of the next polypeptide chain, whereas the other two-domain GH family 3 enzymes form the site within the same molecule. Another striking feature is that the loops located around the active site show high flexibility. One of the flexible loops contains the general acid–base His173 and was thought to be involved in substrate distortion during catalysis. In addition, a loop in close contact with the active site, which comes from the C-terminal domain of the next polypeptide chain, contains a region of high B-factor values, indicating the possibility that the C-terminal domain is involved in catalysis. These results suggest that the dimer structure of NagA is important for its activity and thermostability. Database Structural data are available in the Protein Data Bank under accession number 3WO8. Structured digital abstract ● NagA and NagA bind by x-ray crystallography (View interaction)

Abbreviations (GlcNAc)2, N,N0 -diacetylchitobiose; BsNagZ, b-N-acetylglucosaminidase from Bacillus subtilis; GH, glycoside hydrolase; GlcNAc, b-Nacetylglucosamine; MAD, multi-wavelength anomalous diffraction; MurNAc, N-acetylmuramic acid; NagA, b-N-acetylglucosaminidase from Thermotoga maritima; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate.

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Introduction Chitin, an insoluble polysaccharide of b-1,4-linked Nacetylglucosamine (GlcNAc), is a common constituent of fungal cell walls, the exoskeletons of insects and the shells of crustaceans. GlcNAc and its oligosaccharides derived from hydrolysis of chitin have a variety of biological functions and are used as food additives and medicines. In nature, chitin is hydrolyzed into diacetylchitobiose (GlcNAc)2 by a combination of endo- and exo-type chitinases, followed by dimer processing with b-N-acetylglucosaminidase. According to the CAZy database [1], b-N-acetylglucosaminidases (EC 3.2.1.52) belong to a large group of glycoside hydrolases (GH) family 3, which includes bglucosidases (EC 3.2.1.21) and exo-1,3-1,4-glucanases (EC 3.2.1.-), among others. NagZ is a b-N-acetylglucosaminidase, found within Gram-negative and Gram-positive bacteria, that acts in the peptidoglycan recycling pathway to remove the non-reducing end GlcNAc residue from peptidoglycan fragments [2,3]. Until now, only four structures have been determined for NagZ. Interestingly, NagZs are divided into two groups according to size: the smaller enzymes have a single domain, while the larger enzymes consist of two domains. The NagZs for which structures are known are single-domain NagZs from Gram-negative bacteria Vibrio cholerae (VcNagZ) [4], Salmonella typhimurium (StNagZ) [5] and Burkholderia cenocepacia (BcNagZ) [6], and two-domain NagZs from Gram-positive bacteria Bacillus subtilis (BsNagZ) [5,7]. VcNagZ, StNagZ and BcNagZ adopt a TIM-barrel fold, which is equivalent to that of the first domain (the N-terminal domain) of BsNagZ. Thermotoga maritima is a Gram-negative hyperthermophilic, anaerobic and fermentative saccharolytic bacterium, catabolizing diverse carbohydrates. Although chitinase genes have not been found in the genome of Thermotoga sp., b-N-acetylglucosaminidase (NagA) (EC 3.2.1.52) has been identified in T. maritima [8]. NagA belongs to GH family 3 and shows highly thermostability and hydrolyzes chitooligomers, such as (GlcNAc)2 or (GlcNAc)3, into monomer GlcNAc. GH family 3 b-N-acetylglucosaminidases contain the highly conserved sequence motif KH(F/I)PG(H/L)GxxxxD(S/ T)H, which includes the putative N-acetyl group binding region [9]. NagA is supposed to consist of two domains, and the conserved sequence motif is located at its N-terminal domain [8]. Although the N-terminal domain of NagA shows high sequence identity to those of other GH family 3 b-N-acetylglucosaminidases, the C-terminal domain does not share sequence identity with those of other GH family 3 enzymes, and the role of this unu-


Crystal structure of b-N-acetylglucosaminidase

sual domain remains unclear. NagA hydrolyzes the chitin oligosaccharides and liberates the non-reducing end GlcNAc, as well as other b-N-acetylglucosaminidases; however, NagA is distinct from other GH family 3 b-Nacetylglucosaminidases in that NagA shows high activity against (GlcNAc)2 [8]. Furthermore, biological characterization has revealed that NagA exists as a homodimer [8], while the other GH family 3 enzymes with known structures are monomeric. Thus, NagA has been expected to have a unique three-dimensional structure that does not resemble other GH family 3 enzymes. In this paper, we present the structure of NagA, the first structure of a dimeric two-domain b-N-acetylglucosaminidase from the GH family 3 enzymes. Comparing the structure with that of BsNagZ, whose structure and catalytic mechanism have been well characterized [7], provided insight into the homodimer organization concerned with the active site region, allowing us to speculate on the role of the unusual C-terminal domain. Results Crystallography Purified NagA solution was subjected to screening for crystallization conditions. With the optimized reservoir conditions described in Materials and methods, crystals of NagA appeared after 1 week. Crystal structure analysis revealed that crystals grew with symmetry consistent with space group P3121 or P3221 under the crystallization conditions. Data collection and refinement statistics are summarized in Table 1. The structure of NagA was not solved by molecular replacement with BALBES [10]. Instead, it was solved by the multi-wavelength anomalous diffraction (MAD) method. The correct space group was identified as P3121 during phasing. Two molecules of NagA per asymmetric unit gave a crystal volume per protein mass (VM) [11] of 3Da1 and a solvent content of 65.5% (v/v). 3.56 A The crystal structure of NagA was determined to resolution with Rcryst and Rfree values of 19.3% 2.43 A and 24.6%, respectively. In the asymmetric unit, two polypeptides (Mol-A, Mol-B) correlated with a noncrystallographic two-fold axis were observed. The dimer assembly is consistent with the data from gel-filtration chromatography [8], suggesting that the crystal structure of NagA in the asymmetric unit corresponds to the biologically active dimer form of the protein. Overall structure of NagA From the crystal structure analysis, it was clarified that NagA forms an active dimer, and the monomer

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Table 1. Data collection and refinement statistics. Rmerge = ΣjΣh |Ih,j – 〈Ih〉|/ΣjΣh 〈Ih〉. Rcryst = Σ||Fo| – ||Fc||/Σ|Fo| calculated from 95% of the data, which were used during the course of the refinement. Rfree = Σ||Fo| – ||Fc||/Σ|Fo| calculated from 5% of the data, which were used during the course of the refinement. Values in parentheses are for the highest resolution shell.

Data collection X-ray source Detector Wavelength ( A) Space group Unit-cell parameters ( A) Resolution range ( A) Total no. of reflections No. of unique reflections I/r(I) Redundancy Completeness (%) Rmerge (%) Refinement statistics Resolution ( A) No. of reflections Rcryst/Rfree (%) Mean B value ( A2) No. of non-H atoms rmsd from ideal values Bond lengths ( A)/angles (deg.) Ramachandran statistics (%) Favored Allowed

NagA native

Peak

Edge

Remote

SPring-8 BL38B1 ADSC Quantum 210r 1.00000 P3121 a = b = 133.46, c = 142.33 50.00–2.43 (2.52–2.43) 381 920 54 833 16.0 (2.9) 7.0 (4.0) 98.6 (96.6) 9.5 (32.8)




26.56–2.43 48 565 19.4/24.6 51.2 7196 0.014/1.736

95.2 99.9

structure of NagA consists of two distinct domains, the N- and C-terminal domains, which are connected by an a14 helix (residues 322–332) (Fig. 1A). The N-terminal domain (residues 1–321) constitutes a TIM-barrel fold, which is a common structure among glycoside hydrolases. The C-terminal domain (residues 333–467) displays an aba-sandwich fold, which comprises six b-strands with two a-helices on either side of the sheet. The first 14 residues, including a His-tag sequence, and the nine C-terminal residues were not visible in electron density maps. The dimer structure of NagA revealed that the Nterminal domain of Mol-A makes extensive interaction with the C-terminal domain of Mol-B and vice versa (Fig. 1B). The buried surface area upon dimer forma2 and a corresponding solvation free tion is 4890 A energy gain of approximately 26.9 kcalmol1 was calculated using the program PISA [12]. These values indicate that both monomers are intimately associated and are significantly stabilized upon dimer formation [12]. In both molecules, two loop regions (residues 121–127 and residues 171–174) were not modeled 5094

because of poor electron densities (Fig. 1C). This implies high structural flexibility of these loop regions. Furthermore, the loop-connected b8-strand and a15helix (hereafter named the ‘protrusion loop’) of the Cterminal domain from the next polypeptide comes into close contact with the cavity of the N-terminal domain (Fig. 1C). As discussed later, these loops are located around the active site region. Active site As described above, BsNagZ is a b-N-acetylglucosaminidase whose structure and catalytic mechanism have been discussed in detail [7]. GH family 3 b-Nacetylglucosaminidases use a two-step double displacement mechanism in which two active site residues assist the formation and breakdown of a covalent glycosyl-enzyme intermediate [9,13]. In BsNagZ, Asp318 and His234 were identified as the catalytic nucleophile and the general acid–base catalyst, respectively [7]. His234 further hydrogen bonded to Asp232, and this Asp-His dyad was critical for catalytic activity [7]. As FEBS Journal 281 (2014) 5092–5103 ª 2014 FEBS

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A

B

Fig. 1. Crystal structure of NagA as a ribbon model. The N- and C-terminal domains of Mol-A are denoted N and C, respectively. The domains of Mol-B are denoted N0 and C0 in the same way. (A) Monomer structure of NagA, with different colors for a-helix (red), b-strand (yellow) and loops (green). (B) Dimer structure of NagA in the asymmetric unit. Protein monomers (Mol-A and Mol-B) are colored blue and magenta, respectively. The structure of Mol-A is drawn in the same orientation as the left structure in (A). The cavity of the N-terminal domain is boxed. For clarity, the cavity is shown only for Mol-A, but Mol-B has a similar cavity. (C) A close-up view of the box in (B). The side chains of the conserved motif KHFPGHG and the putative nucleophile Asp245 are displayed as sticks (green). The undefined electron density map (Fo – Fc) at 3r in the cavity is shown in dark blue. All structural figures were drawn with PYMOL [27].

C

shown by the sequence identity between NagA and BsNagZ in Fig. 2, these residues are well conserved in NagA as Asp245 and the Asp171-His173 dyad, respectively. To confirm the role of these residues in the catalytic activity, we prepared mutant enzymes in which


amino acids Asp245, Asp171 and His173 were changed to Ala (D245A, D171A and H173A) and examined their activity using NMR experiments. The analysis of the 1H NMR spectra of (GlcNAc)2 indicated that wild-type NagA completely hydrolyzed the substrate

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into GlcNAc; however, D245A, D171A and H173A mutants had no activity (Fig. 3). The results indicated that the three conserved residues are important for maximal catalytic activity and should play an essential role in catalysis. Interestingly, the electron density of the loop structure containing Asp171 and His173 was not observed in the crystal structure. This loop, which is named the ‘catalytic loop’ [5], is flexible and seems to be important for the substrate recognition and induced fit mechanism. The nucleophilic residue (Asp245) was located in the cavity of the N-terminal domain. The depth of the cleft exhibits enough space to bind the substrate (GlcNAc)2. Indeed, secure electron densities have been observed in a cavity formed by the N-terminal domain (Fig. 1C). The electron density map may be assigned to glycerol from the cryoprotectant solution and phosphate ion derived from the reservoir solution for crystallization or elsewhere. When water molecules were added to the observed electron density map and the structure was refined, the calculated B-factor value 2. Moreover, Fo Fc density was still was 26.9 A observed at 3r. Therefore, the possibility that the electron density was attributable to water molecules was excluded. However, the resolution is not sufficient to identify the molecule in the cavity; for example, the exact positions of four oxygen atoms surrounding the central phosphorus atom in the phosphate cannot be resolved. Anyway, it is suggested that the active site cleft could allow the substrate to access the catalytic site easily. In order to obtain more precise information regarding the catalytic loop, we also attempted co-crystallization of NagA with the inhibitor O-(2-acetamido-2-deoxy-Dglucopyranosylidene) amino N-phenylcarbamate (PUGNAc) [14]. Despite numerous crystallization trials using various inhibitor concentrations, we were unable to observe the complex structures bound with the inhibitor. Detailed experiments designed to identify the complex structure are in progress.

the N-terminal domain of these enzymes. The sequence identities of their N-terminal domains are estimated to be 29.4% (residues 4–319 for NagA and 42–396 for BsNgaZ). Thus, we compared the monomer structure of NagA (Mol-A) with that of BsNagZ (Protein Data Bank entry 3BMX) by superimposing their N-terminal domains. The structures of both N-terminal domains however, coincide well, with an rmsd value of 1.54 A; the C-terminal domains did not superimpose at all (Fig. 4A). As shown in Fig. 4A, the C-terminal domains deviated perpendicularly, indicating that the relative position between the N- and C-terminal domains is quite different in these enzymes. The position of the NagA domains is mainly caused by the dimer interaction; however, the domain orientation is also restricted by the length of the domain linker. As shown in Figs 2 and 4A, the N- and C-terminal domains are joined with a14 and a20–21 for NagA and BsNagZ, respectively. The length of a14 of NagA whereas that of a20–21 of is approximately 20 A, Therefore, the length BsNagZ is approximately 40 A. shorter of the linker in NagA is approximately 20 A than that in BsNagZ, which would not allow NagA to form the same structure as that of BsNagZ. Although both C-terminal domains exhibit the aba-sandwich fold, the sequence identity between them is very low (4.76%) and the rmsd between the structures (333–458 for NagA, 435–605 for BsNagZ) was calculated as However, it is of great interest that the 3.35 A. arrangement and position of the C-terminal domain in BsNagZ are almost the same as those of the C-terminal domain of NagA (Mol-B) (Fig. 4B). The active site cleft of BsNagZ is formed by two domains in the same molecule [7], but that of NagA is formed by two domains in different molecules. This difference in formation of the active site cleft caused changes in the shape and size of the active site, which may affect the catalytic mechanism and substrate specificity.

Structural comparison with related b-Nacetylglucosaminidase

Substrate-binding site

A structural homology search of NagA using the DALI server [15] identified several enzymes in GH family 3. As expected, the highest similarity was assigned to BsNagZ (Z score of 41.0). Although both NagA and BsNagZ consist of two domains, their biological assembly differs. As described above, the catalytic nucleophile of the aspartate residue and the conserved motif KH(F/I)PG(H/L)GxxxxD(S/T)H, which contains the general acid–base Asp-His dyad (in bold), lies on 5096

Discussion

The crystal structures of BsNagZ in complex with the inhibitor PUGNAc or the disaccharide substrate GlcNAc-N-acetylmuramic acid GlcNAc-MurNAc have provided some informative insights into the catalytic mechanism of b-N-acetylglucosaminidase [5,7]. The inhibitor or the substrate was observed in the cavity of the N-terminal domain, suggesting that the C-terminal domain does not participate in catalysis, although BsNagZ is a two-domain enzyme with a monomer structure. BsNagZ binds the disaccharide substrate FEBS Journal 281 (2014) 5092–5103 ª 2014 FEBS

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Fig. 2. Sequence alignment of NagA and BsNagZ. Identical positions are marked with asterisks below the alignment. The secondary structure above the alignment is for NagA. Residues belonging to helices and b-strands are indicated red and blue, respectively. The conserved motif KHFPGHGxxxxDSH is boxed. The domain linker is boxed with a dashed line and a20 and a21 of BsNagZ are shown below the alignment. The catalytic nucleophile Asp and the general acid–base Asp-His dyad are shown as white letters on a black background.

GlcNAc-MurNAc in both a distorted (PDB entry 4GYJ) and a relaxed (PDB entry 4GYK) conformation [5]. Superimposition of the structure of the N-terminal domain of NagA onto these substrate-bound structures of BsNagZ allowed prediction of the catalytic and substrate-binding residues of NagA. The active site of GH family 3 NagZ consists of two glucosyl-binding subsites (1 and +1) with a catalytic nucleophile and general acid–base residue [5]. The 1 and +1 subsites recognize the non-reducing and reducing end, respectively, with the cleavage occurring between the 1 and +1 subsite [16]. In the 1 subsite of BsNagZ, Asp123, Arg191, Lys221 and His222 bind to the non-reducing end-terminal GlcNAc sugar [5,7], and these residues are strictly conserved in NagA as Asp63, Arg130, Lys160 and His161, respectively (Fig. 5A,B). Asp245 of NagA is located in the same position as Asp318 (Asn318),

the catalytic nucleophile of BsNagZ (Fig. 5A,B). These observations, combined with our mutagenesis analyses, suggest that the substrate recognition mode in the 1 subsite is probably identical for these enzymes. This is also supported by the fact that the observed electron densities in the 1 subsite of NagA (which is illustrated in Fig. 1C) superimposed well on GlcNAc (Fig. 5A,B) because the non-reducing end of the natural substrate of both enzymes is GlcNAc sugar. Although structural similarities are present in the 1 subsite of NagA and BsNagZ, the +1 subsite seems to be different because the reducing ends of the substrates are GlcNAc and MurNAc, respectively. However, MurNAc is a derivative of GlcNAc, and so it must be reasonable to predict the substrate-binding residues of NagA using the structure of GlcNAc-MurNAc-bound BsNagZ. In BsNagZ, Arg57, which is highly conserved in NagZ, appeared to interact with

A

B

Fig. 3. Activity of wild-type and mutant NagA. (A) The reaction scheme of NagA with (GlcNAc)2 as a substrate. (B) A methyl proton region of the 1N NMR spectrum of (GlcNAc)2 after incubation with NagA for 120 min at 25 °C. I, the Nacetyl group of the non-reducing end of (GlcNAc)2; II, the N-acetyl group of the reducing end of (GlcNAc)2; III, the N-acetyl group of GlcNAc. These chemical shifts were assigned previously [28].

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A


B

Fig. 4. Structure comparison of NagA with BsNagZ. Comparison was performed by superimposing their N-terminal domains. (A) Superposition of the monomer structures of NagA (blue) and BsNagZ (yellow) (PDB entry 3BMX). The domain linkers of the helixes are colored red. (B) Superposition of the dimer structure of NagA and BsNagZ. The N-terminal domain of Mol-A and the C-terminal domain of Mol-B of NagA are colored blue and magenta, respectively.

acetamido group of MurNAc through hydrogen bonding [5], whereas the corresponding residue of Asn14 in NagA is located too far from the substrate to interact with it (Fig. 5A,B). The most curious thing is that Ser358 in the C-terminal domain of NagA from the next polypeptide comes close to the substrate (Fig. 5A, B). Ser358 of NagA is not conserved in NagZ but is located on the protrusion loop and has higher B-factor values (Fig. 6), suggesting that Ser358 could be positioned within the active site so that it will interact with the acetamido group of GlcNAc. This also means that the C-terminal domain of NagA may participate in substrate recognition, whereas BsNagZ does not. Analysis of the substrate-bound NagA structure is in progress. Catalytic loop According to the mechanism of BsNagZ [7], the following catalytic mechanism of NagA is proposed. The general acid–base catalytic His173 hydrogen bonds with Asp171, allowing protonation of the oxygen of the glycosidic bond and facilitating removal of the leaving group by nucleophilic attack of Asp245. Then His173 promotes breakdown of the covalent glycosylenzyme intermediate by removing a proton from an incoming water molecule. The catalytic nucleophile of aspartic acid is strictly conserved among all known members of GH family 3 [9]. However, the catalytic mechanism involving a histidine residue is unusual, because a glutamate or aspartate residue is used as the general acid–base in most glycoside hydrolases [17]. As FEBS Journal 281 (2014) 5092–5103 ª 2014 FEBS

described above, the catalytic loop containing the Asp171-His173 dyad of NagA was too disordered to build an unambiguous model (Fig. 1C). The substrate– complex structures of BsNagZ and StNagZ also revealed that the corresponding catalytic loop undergoes significant structural changes during catalysis [5]. A superposition of the structures of BsNagZ with substrate revealed that the conformation of the catalytic loop varied between these structures. In the substrate distorted conformation (PDB entry 4GYJ), the catalytic loop was positioned within the active site such that His234 (Ne2) was allowed to interact with the glycosidic oxygen of the substrate (Fig. 5A). In contrast, the substrate relaxed conformation (PDB entry 4GYK) revealed that the catalytic loop was moved outwards from the active site, similar to NagA, placing away (Fig. 5B). These difHis234 (Ne2) about 21.9 A ferences in the structure of the catalytic loop are supposed to be correlated with the conformation of the substrate during the catalytic process. The disorder conformation of the catalytic loop allows the proposed general acid–base residue to reposition and play a role in substrate distortion during catalysis [5]. Taken together with our results, these observations suggest that catalytic loop mobility is a common feature in GH family 3 b-N-acetylglucosaminidase despite differing preferences for the sugar moiety of the substrates. Interestingly, the other loop (residues 121–127) around the active site was also disordered because of its flexibility (Figs 1C and 5A,B). Furthermore, the protrusion loop from the C-terminal domain of the next polypeptide, especially the residues Glu355–

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A

B

Fig. 5. Stereoview of the active site of NagA and BsNagZ. Superposition of the active site and catalytic loop of NagA and substrate-bound BsNagZ are shown. BsNagZ bound to the substrate of distorted (PDB entry 4GYJ) (A) and relaxed conformation (PDB entry 4GYK) (B). In both BsNagZ, the catalytic nucleophile Asp318 was mutated to Asn (Asn318). Yellow sticks represent the disaccharide substrate GlcNAc-MurNAc. Cyan sticks with blue labels are residues of BsNagZ. The corresponding residues of NagA are shown in gray with black labels. The catalytic loops of BsNagZ and NagA are also shown in cyan and gray, respectively. The next polypeptide chain (Mol-B) of NagA is colored magenta. The undefined electron density map (Fo – Fc) of NagA in Fig. 1C is shown in green.

Asn356, which are close to the active site, have higher B-factor values (Fig. 6). Thus, in a similar manner to the catalytic loop, flexibility of these loops may be related to substrate distortion during catalysis. In the structure of BsNagZ, however, no part of the C-terminal domain comes into close contact with the active site on the N-terminal domain (Fig. 6). These results indicate that NagA possesses a more closely packed environment in the active site compared with BsNagZ, and the roles of the C-terminal domain are quite dif5100

ferent in these enzymes, even though they are both two-domain GH family 3 b-N-acetylglucosaminidases.

Conclusion The first crystal structure of NagA was solved at a res NagA forms a dimer structure in olution of 2.43 A. which the monomer structure consists of an N- and a C-terminal domain. The active site cleft is formed by the N-terminal domain and the C-terminal domain FEBS Journal 281 (2014) 5092–5103 ª 2014 FEBS

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Fig. 6. The relation between the active site and the C-terminal domain. The surface models of the N-terminal domains are shown in light gray. The structures of the C-terminal domains are colored red (high B-factors) to blue (low B-factors). The flexibility loops around the active site are boxed with dashed lines. The catalytic nucleophilic Asp residues are colored red. The two panels are drawn in the same orientation as the left structure in Fig. 4B.

from the next polypeptide chain. However, the active form of BsNagZ is a monomer structure. The result that the position of the C-terminal domain in BsNagZ is almost the same as that of the C-terminal domain of NagA (Mol-B) (Fig. 4B) suggests that the dimer structure of NagA is necessary for its activity. The active site cleft of BsNagZ is formed by two domains in the same molecule, but that of NagA was formed by two domains in different molecules. The buried surface 2, with area upon dimer formation of NagA is 4890 A a corresponding high solvation free energy gain of approximately 26.9 kcalmol1. These values indicate that both monomers are intimately associated and form a significant stable dimer structure. The dimer structure of NagA may be important for its activity and thermostability.

Materials and methods Protein expression and purification The genomic DNA of T. maritima strain MSB8 (NBRC 100826G) was obtained from the National Institute of Technology and Evaluation (Chiba, Japan). The nagA gene (residues 1–467; GenBank accession no. FJ172673.1) was amplified from the genomic DNA of T. maritima MSB8 by PCR using primers with NdeI and BamHI. The amplified DNA was digested with NdeI and BamHI and then ligated with the corresponding sites in the expression vector pCold-II (Takara Bio, Shiga, Japan). Thus, the recombinant NagA was fused with 11 extra amino acid residues containing a six-residue histidine tag at its N-terminus. Mutants of NagA, in which Asp171, His173 or


Asp245 were replaced by Ala (D171A, H173A and D245A, respectively), were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Escherichia coli Rosetta (DE3) pLysS was transformed with the resultant plasmids harboring nagA genes, and the transformed cells were grown at 37 °C in LB medium. When the cell density reached an A600 nm of 0.5, the culture was shifted to 15 °C to induce protein expression. The cells were harvested at 16 h after induction and disrupted by sonication in 30 mL buffer A (50 mM Tris/HCl, pH 8.0). The cell debris and insoluble proteins were removed by centrifugation at 15 000 g for 30 min after heat treatment at 80 °C for 30 min. The supernatant was loaded onto a HisTrap HP (GE Healthcare, Uppsala, Sweden) column equilibrated with buffer A, and the bound protein was eluted with buffer A containing 0.5 M imidazole. The protein solution was dialyzed against buffer A and applied onto a HiTrap Q HP (GE Healthcare) column. The proteins were eluted with a linear gradient of 0–1.0 M NaCl, and the peak fractions containing NagA were pooled and loaded onto a HiLoad 26/600 Superdex 200 prep-grade column (GE Healthcare) equilibrated with 20 mM Tris/HCl, pH 8.0, containing 150 mM NaCl. Selenomethionine-substituted NagA for the MAD method was prepared following the procedure described above, except that the cells were grown in LeMaster broth [18].

Crystallography Crystals of NagA were obtained by the hanging drop vapor-diffusion method. A mixture consisting of 1.5 lL

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protein solution (10.0 mgmL1) and an equal volume of the reservoir solution (0.4 M ammonium phosphate) was equilibrated against the reservoir solution at 20 °C. Crystals were immersed in a cryoprotectant solution consisting of reservoir solution supplemented with 25% (v/v) glycerol and then flash-cooled in liquid nitrogen at 100 K. X-ray diffraction data were collected at synchrotron beamline BL38B1 (SPring-8, Hyogo, Japan). Data were processed and scaled using the HKL-2000 program suite [19], and 180 (native, edge, remote) and 360 (peak) images with 1° oscillation were collected. Exposure time and crystal-todetector distance were 6 s (native) and 4 s, and 240 mm (native) or 250 mm (peak, edge, remote), respectively. The initial phase was calculated with SOLVE [20], and a model was built with BUCCANEER [21]. Further model building was performed with COOT [22], and the structure was refined with REFMAC [23] and CNS [24] by rigid body refinement, simulated annealing refinement, positional minimization, water molecule identification, and individual isotropic Bfactor value refinement. The structure was validated by MOLPROBITY [25].

Enzymatic activity of NagA mutants The hydrolase activities of NagA mutants were detected using 1H NMR spectroscopy. NMR experiments were conducted at 25 °C on a Varian Inova (600 MHz) equipped with a z-gradient, triple-resonance TR probe. The chemical shifts were referenced to internal 4,4-dimethyl-4-silapentane-1-sulfonic acid. The substrate analyte (GlcNAc)2 was prepared at 1.2 mM in 50 mM potassium phosphate buffer (pH 6.0) containing 10% D2O (w/w) for the NMR experiments. The reaction was initiated by adding wild-type NagA or mutants to a final concentration of 1 lM. The water signal was suppressed using the Watergate pulse sequence [26]. The one-dimensional 1H signals consisted of 8192 sampling points covering a spectral width of 15 ppm. The relaxation delay was set at 1 s, and 64 scans were accumulated for each spectrum. This acquisition was repeated every 1.45 s.

Acknowledgements The authors are grateful to Dr T. Nakamura (AIST) for diffraction data collection of NagA crystals. The authors are grateful to Drs Y. Yoshida (AIST) and T. Sato (AIST) for their support throughout these studies. This work was supported in part by a Grant-inAid for Scientific Research (Grant Number 25450143) to S. Mine from the Japan Society for the Promotion of Sciences. Y. Kawarabayasi was partially supported by the Institute for Fermentation, Osaka (IFO). Synchrotron experiments were performed at SPring-8 under the approval of the Japan Synchrotron Radiation

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Research Institute (Proposal nos. 2012B1098 and 2014A6953).

Author contributions SM planned the experiments, performed the experiments, analyzed the data and wrote the paper. Y Kado, MW and KI analyzed data. YF, YA, TU, Y Kawarabayasi and TI performed experiments.

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