Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Masahiro Nakajima; Ryuta Yoshida; Akimasa Miyanaga; Koichi Abe; Yuta Takahashi; Naohisa Sugimoto; Hiroyuki Toyoizumi; Hiroyuki Nakai; Motomitsu Kitaoka; Hayao Taguchi

doi:10.1371/journal.pone.0148870

Abstract

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.

Citation: Nakajima M, Yoshida R, Miyanaga A, Abe K, Takahashi Y, Sugimoto N, et al. (2016) Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua. PLoS ONE 11(2): e0148870. https://doi.org/10.1371/journal.pone.0148870

Editor: Israel Silman, Weizmann Institute of Science, ISRAEL

Received: December 6, 2015; Accepted: January 25, 2016; Published: February 17, 2016

Copyright: © 2016 Nakajima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All coordinate files are available from RCSB Protein Data Bank (PDB IDs 4ZO6, 4ZO7, 4ZO8, 4ZO9, 4ZOA, 4ZOB, 4ZOC, 4ZOD, and 4ZOE).

Funding: The authors have no support or funding to report.

Competing interests: The authors have declared that no competing interests exist.

Introduction

β-1,2-Glucan is a homo-polymer composed of glucose, as are cellulose (β-1,4-glucan) and laminarin (β-1,3-glucan), and is found mainly in some Gram-negative bacteria, such as Agrobacterium, Rhizobium, Shinorhizobium, and Brucella, as a cyclic form [1–5]. Cyclic β-1,2-glucan is known as an infectious or commensal factor in animals or plants and is used as a modulator of intracellular osmotic pressure after secretion into the extracellular space and accumulation in the periplasm [4–7]. Linear β-1,2-glucan is also found as an extracellular or periplasmic glucan with degrees of polymerization (DP) of 5–13 possessing β-1,6-glucosyl branches in Escherichia coli and Pseudomonas syringae [8–10]. In addition, sophorosides are found in some plants [11]. In contrast to the existence of β-1,2-glucan in nature, only a few β-1,2-glucan degrading enzymes has been reported. Though β-1,2-glucan-degrading glucanases and glucosidases induced by β-1,2-glucan have been reported in Cytophaga (Chitinophaga) arvensicola, a Gram negative bacterium, and Acremonium sp. 15, a filamentous anamorphic fungus [12,13], their amino acid sequences have not been elucidated.

Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase (OGP), an enzyme specific to β-1,2-glucan. The cytosolic Lin1839 enzyme catalyzes reversible phosphorolysis of β-1,2-glucans with DP of 3 or more to produce α-glucose 1-phosphate (G1P) [14], but the enzyme does not act on sophorose (Glc-β-1,2-Glc, Sop₂). Thus, it is likely that other enzymes are also required for complete dissimilation of β-1,2-glucans. In the gene cluster containing the lin1839 gene, the lin1840 gene encodes a putative glycoside hydrolase family (GH) 3 β-glucosidase (BGL) (GenBank accession number: CAC97071.1) [15]. These facts led us to hypothesize that the Lin1840 protein is suitable for Sop₂ degradation, although there is no experimental evidence supporting this hypothesis.

GH3 is one of the major families containing BGLs along with GH1. BGLs form a large subgroup widely distributed in animals, plants, and microorganisms in GH3 containing N-acetyl-β-d-glucosaminidases, α-l-arabinofuranosidases and β-d-xylopyranosidases as well [16]. The substrate recognition residues and their structural positions for non-reducing end glucosides are highly conserved among GH3 BGLs. On the other hand, the substrate recognition sites in other moieties of substrates exhibit great diversification in the family, which leads to a variety of substrate and chain length specificities [17].

Lin1840 forms a clade with closely related homologs phylogenetically [18]. Only two enzymes have been characterized and/or are structurally available in the clade. Metagenomic GH3 β-glucosidase from compost (JMB19063), which is the only structurally available enzyme, was reported to act on cellooligosaccharides (β-1,4-glucooligosaccharides, Cel_ns) [18], and Flavobacterium meningosepticum GH3 β-glucosidase (FmBGL) has been identified as an aryl β-glucosidase [19]. The amino acid sequence identities of these two enzymes with Lin1840 are 42% and 40%, respectively. Arg587, which is one of the main residues comprising subsite +1 in JMB19063, is highly conserved among the closely related homologs (S1 Fig). The presence of a conserved arginine residue important for substrate recognition led us to expect a similar function among Lin1840, JMB19063, and FmBGL. Cel_ns were the only natural compounds tested as substrates for previous characterization of JMB19063 and FmBGL. However, both enzymes have very low activity on Cel_ns, whereas they show high activity toward p-nitrophenyl-β-d-glucopyranoside (pNP-β-Glc), an artificial substrate [18,19]. This means that previous reports of FmBGL and JMB19063 characterization lack information supporting or denying the hypothesis that Lin1840 is involved in β-1,2-glucan metabolism in L. innocua. In this study, we describe the characteristics and structure-function relationship of the Lin1840 to provide the first mechanistic insight into recognition of β-1,2-glucooligosaccharides (Sop_ns) by BGLs.

Materials and Methods

Preparation of recombinant Lin1840 and mutants

Gene cloning and overexpression of lin1840 from L. innocua Clip11262 and purification of recombinant Lin1840 (Lin1840r) was described in our previous paper [20]. Briefly, the protein fused with a C-terminal His₆-tag was purified from the cell extract of the transformant using a HisTrap FF crude column (5 ml; GE Healthcare, Buckinghamshire, England), and then was buffered with 50 mM 3-(N-morpholino)propanesulfonic acid (Mops) buffer (pH 7.0) using Amicon Ultra 30,000 molecular weight cut-off (Millipore, Billerica, MA, USA) for enzyme assay. The protein was further purified using Superdex 200 (Hiload 16/60; GE Healthcare) for crystallization. The molecular weight of Lin1840r in solution was estimated from the retention time. Ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa; GE Healthcare) were used as standard proteins. Blue dextran 2000 (2000 kDa; GE Healthcare) was used to determine the void volume of the column. Protein concentration was determined by UV absorbance at 280 nm (extinct coefficient [21] and theoretical molecular weight of Lin1840r are 67770 cm⁻¹M⁻¹ and 80532.2 Da, respectively). Construction of plasmids for expression of the D270A, E473A, and R572K mutants was performed based on the protocol for a KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan) using pET-28a inserted with lin1840 as a template, KOD Plus (TOYOBO, Osaka, Japan), and the primers described below. The primer pairs used for amplification of the D270A, E473A, and R572K mutant genes were 5’-TGGGGCGCTGTTGCCGAAGTAATTAATCAC-3’ and 5’-CGCAGAAATAAGTACACCGTCAAACTCCA-3’, 5’-CCCGCCCCATTCATTTTTTTCACCTAGCGC-3’ and 5’-GCGGCAGGAAGTCTTGCTACTATTCG-3’, and 5’-GAGCGCCACAAACACCGGAAAATAAAGG-3’ and 5’-CAGTGCGTAAATGATTATAATAAACTGG-3’ (mutated nucleotides are underlined), respectively. Production and purification of the mutant enzymes were performed in the same way as for the WT.

Enzyme assays using p-nitrophenyl-sugars

The substrate specificity of Lin1840r toward pNP-β-Glc, pNP-β-d-xylopyranoside, pNP-β-d-fucopyranoside, pNP-β-d-arabinofuranoside, and pNP-β-d-galactopyranoside (Sigma Aldrich, St. Louis, MO, USA) was tested. Glycerol was used for stabilization of Lin1840r during the reaction. The reaction mixture comprising 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (pH 6.0), 5 mM substrate, 15% glycerol and Lin1840r in a total volume (20 μl) was incubated at 20°C for 10 min. After incubation, 10 μl of the reaction mixture was taken and mixed with 90 μl of 0.2 M Na₂CO₃ in a 96-well plate (EIA/RIA, plate, 96-well half area; Corning, NY, USA) to stop the reaction. The hydrolytic activity toward pNP-sugars was determined by measuring the absorbance at 405 nm derived from free pNP using a microplate reader (SpectraMax190; Molecular Devices, CA, USA). Various concentrations of pNP (0.005–0.5 mM) were used as standards.

Kinetic analysis using pNP-β-Glc and β-linked gluco-oligosaccharides

To determine the kinetic parameters of pNP-β-Glc hydrolysis, 70 μl of a reaction mixture comprising 0.5–30 mM pNP-β-Glc, 15% glycerol and 0.42 μg of the enzyme in 50 mM Mes buffer (pH 6.0) was incubated at 20°C for 10 min. Ten μl aliquots of the reaction mixture were taken and then was mixed with 90 μl of 0.2 M Na₂CO₃ every 2 min to stop the reaction. Activity was determined as described above. The kinetic parameters were determined by non-linear regression of the data using an equation expressing substrate inhibition: v/[E₀] = k_cat[S]/(K_m+[S]+[S]²/K_is) (equation 1), where v is the initial velocity of pNP release, [E₀] the enzyme concentration, and K_is the substrate inhibition constant. The kinetic analysis regarding β-linked gluco-oligosaccharides was performed using Sop_ns [14], β-1,2-glucans (average DP 25) [22], Lam_ns (BIOCON (JAPAN) LTD, Aichi, Japan), Cel₂ (Wako Pure Chemical Industries, Osaka, Japan), and Gen₂ (Tokyo Chemical Industry, Tokyo, Japan) as substrates. The reaction mixtures (70 μl) comprising various concentrations of substrates, the enzyme, and 15% glycerol in 50 mM Mes buffer (pH 6.0) were incubated at 20°C for 10 min. At intervals of two minutes, 10 μl aliquots of the mixtures were taken to stop the reaction by heat treatment at 99°C. Then the samples mixed with 90 μl of GOPOD FORMAT KIT (Megazyme, Wicklow, Ireland) were incubated at 45°C for 20 min. When the substrates were Sop_n and Lam_n, 6 μl aliquots of the samples and 114 μl of GOPOD FORMAT KIT were mixed. The amounts of Glc released from oligosaccharides were calculated from absorbance at the 510 nm using 100 μl aliquots of the solutions. The concentrations of Glc released from disaccharides were taken to be half since two Glc molecules were released in hydrolysis of one substrate molecule. The kinetic parameters were determined by fitting to equation 1 or the normal Michaelis-Menten equation.

Temperature and pH profiles

The reaction conditions and the method for measuring released pNP were the same as described in the kinetic analysis section. The substrate used was 5 mM pNP-β-Glc. The optimum temperature and pH were determined by measuring the activity at various temperatures (0–50°C) and in various pH ranges in the following buffers: sodium acetate (pH 4.0–5.5), Mes (pH 5.5–6.5), Mops (pH 6.5–7.5), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) (pH 7.5–8.5), and N-cyclohexyl-2-aminoethanesulfonic acid (pH 8.5–9.0). The thermal and pH stabilities were determined from the residual hydrolytic activity at 20°C after incubation of Lin1840r (0.1 mg/ml) at various temperatures in 50 mM Mes buffer (pH 6.0) for 1 h, and in 50 mM various buffers with the pH ranges described above at 20°C for 1 h, respectively. The incubated enzyme solutions were diluted at least 20 times with the reaction solution.

Inhibition kinetics

Gluconic acid δ-lactone (GDL) (Wako Pure Chemical Industries), isofagomine d-tartrate (IFG) (Toronto Research Chemicals Inc., Toronto, Canada), and 1-deoxynojirimycin (DNJ) (Wako Pure Chemical Industries) were used as inhibitors. The inhibitory effect on hydrolytic activity toward pNP-β-Glc was measured with various substrates (1.5–10 mM) and inhibitor concentrations in 50 mM Mes buffer (pH 6.0) and 15% glycerol at 20°C. Kinetic parameters were determined using Grafit 7.0.3 by non-linear regression of the data using equations of competitive inhibition: v/[E₀] = k_cat [S]/{K_m (1 + [I]/K_i) + [S]} or mixed-type inhibition: v/[E₀] = k_cat [S]/{K_m (1 + [I]/K_i) + [S] (1 + [I]/K_i’)}, where v is the initial velocity of pNP release, [E₀] the enzyme concentration, K_i the competitive inhibition constant, and K_i’ the noncompetitive inhibition constant.

Crystallography

Crystallization of the WT and D270A mutant was performed by the hanging-drop vapor-diffusion method. As described previously [20], 1 μl of 10 mg/ml Lin1840r in 5 mM Mops (pH 7.0) mixed with 1 μl of reservoir solution comprising 15% (v/v) glycerol, 0.17 M Li₂SO₄, 0.085 M Tris-HCl (pH 9.0), and 25.5% (w/v) PEG4000 was incubated at 25°C for 1 week for crystallization. Crystals were soaked in the reservoir solution supplemented with each ligand for 1 h to obtain complex structures. Crystals of WT were soaked in 150 mM Glc, 5 mM GDL, or 5 mM IFG. D270A crystals were soaked in 100 mM Sop₂, 100 mM Sop₃, 50 mM Lam₂, 300 mM Cel₂, or 300 mM Gen₂. The crystals were cooled and then kept at 100 K in a nitrogen-gas stream during data collection. A set of X-ray diffraction data for the crystal was collected using a CCD detector (ADSC Quantum 210r) on a beamline AR-NW12A at Photon Factory (Tsukuba, Japan). The diffraction data set was processed using iMosflm [23]. A model structure of Lin1840r was predicted using SWISS-MODEL (http://swissmodel.expasy.org/) [24] based on the A chain of HvExoI (PDB code; 1EX1), and then used as a search model for molecular replacement. Molecular replacement was performed using MOLREP [25] to determine initial phases. Automated model building was performed using ARP/wARP [26]. Manual model building and refinement were performed using Coot [27] and Refmac5 [28], respectively. Quality check of the structures was performed using wwPDB validation server (http://wwpdb-validation.wwpdb.org/validservice/). The figures were prepared using PyMOL (DeLano Scientific; http://www.pymol.org). The buried surface area was calculated with the protein-protein interaction interface server (PISA; http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) [29].

Results

General properties of Lin1840r

The amino acid sequence of Lin1840 includes no predicted N-terminal signal peptide, as judged from SignalP 4.0 analysis (http://www.cbs.dtu.dk/services/SignalP/) [30], suggesting that the enzyme is localized in the cytosol. On size-exclusion chromatography, a recombinant Lin1840 protein was eluted as a 150 kDa one, suggesting that it is dimeric. The enzyme did not show significantly decreased catalytic activity on incubation up to 20°C in the pH range of 5.0–9.0, but drastically lost the activity at 40°C. The enzyme exhibited maximal catalytic activity at 37°C and pH 6.0. Asp270 and Glu473 in Lin1840r are predicted to be a catalytic nucleophile and a catalytic acid/base, respectively, according to primary sequence alignment with FmBGL [31,32].

Substrate specificity

The substrate specificity of Lin1840r as to glycone was determined using several p-nitrophenyl (pNP)-β-d-monosaccharides. The enzyme showed no activity toward any of those examined (less than 0.01 U/mg) except for pNP-β-Glc, indicating that it specifically acts upon β-glucosides. The kinetic parameters of the enzyme toward pNP-β-Glc were k_cat = 48 ± 3 (s⁻¹), K_m = 3.1 ± 0.3 (mM), and k_cat/K_m = 15 ± 1 (mM⁻¹ s⁻¹). To investigate the linkage position specificity, kinetic parameters for β-linked gluco-disaccharides were determined (Table 1). Lin1840r showed large k_cat values for Sop₂ and Lam₂, while the k_cat values for Cel₂ and gentiobiose (Glc-β-1,6-Glc, Gen₂) were less than 1% of those for Sop₂ and laminaribiose (Glc-β-1,3-Glc, Lam₂). The K_m values for Sop₂ and Lam₂ were similarly small, while the K_m values for Cel₂ and Gen₂ were approximately 7 and 13 times higher than that for Sop₂, respectively. Consequently, the enzyme exhibited comparable k_cat/K_m values for Sop₂ and Lam₂, while the values for Cel₂ and Gen₂ were both less than 0.05% of that for Sop₂. The kinetic parameters for Sop₂ and Lam₂ were similar to those for pNP-β-Glc. Then kinetic parameters for Sop_ns were determined. The k_cat value for Sop₃ was similar to that for Sop₂, but the k_cat values for Sop₄ and Sop₅ were less than one-tenth of that for Sop₂. The K_m values for Sop_3-5 were 5 or more times higher than that for Sop₂. Consequently, the catalytic efficiency decreased with the increase in DP remarkably. In the case of β-1,3-glucooligosaccharides (Lam_ns), in contrast, the k_cat, K_m or k_cat/K_m value did not markedly change with increasing DP, unlike in the case of Sop_ns. The enzyme did not show significant hydrolytic activity toward β-1,2-glucan (reaction velocity, less than 0.01 U/mg in the presence of 1 mM substrate).

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Table 1. Kinetic parameters of Lin1840r for β-linked gluco-oligosaccharides and pNP-β-Glc.

https://doi.org/10.1371/journal.pone.0148870.t001

Inhibition kinetics

The inhibition modes and constants for six inhibitors as to the hydrolytic activity toward pNP-β-Glc are summarized in Table 2. Three glucosidase inhibitors, GDL, IFG, and DNJ, consistently exhibited competitive-type inhibition with K_i values of less than 1 mM. IFG showed the strongest inhibition among all the inhibitors examined, the K_i value being 4.1 μM. While the K_i value of Lin1840r for IFG was similar to that of Aspergillus aculeatus BGL1 (AaBGL1) (14 μM), the K_i value of Lin1840r for DNJ (290 μM) was over 100 times higher than that of AaBGL1 (2.4 μM) [33]. On the other hand, Glc, Cel₂, and Gen₂ consistently showed mixed-type inhibition (Table 2). The K_i and K_i’ values for Cel₂ were more than 5 times higher than those for Glc and Gen₂.

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Table 2. Inhibition constants for inhibitors.

https://doi.org/10.1371/journal.pone.0148870.t002

Overall structure

The crystal structure of apo wild-type (WT) Lin1840r was determined at 1.8 Å resolution (S1 Table). The structure showed that Lin1840r forms a dimer of identical subunits each composed of three domains (Fig 1A). The structure of Lin1840r was compared with those of two GH3 enzymes, JMB19063 and barley GH3 β-glucan-exohydrolase (HvExoI), exhibiting the highest similarity based on the Z-score with the Dali server (http://ekhidna.biocenter.helsinki.fi/dali_server/) [34] (S2 Table). Lin1840r shows a similar overall structure to that of JMB19063. A loop (amino acids 568–598) in Lin1840r extends into the active site of the other subunit to form a part of the substrate pocket as in the case of the corresponding loop of JMB19063 (Fig 1 and S2A Fig). Therefore, the formation of the dimer is predicted to be important for the catalysis. Contrarily, HvExoI is a monomeric enzyme whose catalytic site is composed of a single subunit [35]. Lin1840r is composed of three domains based on Pfam (http://pfam.xfam.org/) [36]. There are some differences in each domain from those of JMB19063. In domain 1, the N-terminal region of Lin1840r (amino acids 34–53) forms an α helix, whereas it forms a loop in JMB19063 (amino acids 29–48) [18] (S2B Fig). In domain 2, the helix and loop (amino acids 435–455) extending to the active site in JMB19063 are missing in Lin1840r (S2B Fig). In domain 3, clear electron density was observed (S2C Fig). The metal ion obviously undergoes six-coordination with the side chain of Asp648, the backbone carbonyl of Thr650, and four water molecules. The metal ion is suggested to be Mg²⁺ according to the server for checking metal ion binding (http://csgid.org/csgid/metal_sites/, [37]) (S2 Table), although a Mg²⁺ ion was not added to Lin1840r in any step of sample preparation.

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Fig 1. Overall structure of Lin1840r.

(A) Lin1840r is a dimer composed of subunits A and B. Domains 1 ((β/α)₈ triose phosphate isomerase barrel fold, amino acids 1–339), 2 ((α/β)₆ sandwich fold, amino acids 360–543), and 3 (fibronectin type III (FnIII) family, amino acids 605–723) in subunit A are presented in cyan, orange, and magenta, respectively. The loop extending to the active center in subunit B (amino acids 543–604), and the linker region that connects domains 1 and 2 (amino acids 340–359) are shown in red and black, respectively. The corresponding three domains, loop, and linker in subunit B are presented in semitransparent blue, pink, and yellow, brown, and gray, respectively. Mg²⁺ ions are shown as green spheres. N-terminal and C-terminal regions of both subunits are denoted by N and C, respectively.

https://doi.org/10.1371/journal.pone.0148870.g001

Subsite −1 of Lin1840r

The active center of Lin1840r is located at the interface of domains 1 and 2 as in the cases of known GH3 BGLs. The two predicted catalytic residues, Asp270 and Glu473, occupy similar positions to the corresponding Asp residues (catalytic nucleophile) and Glu residues (catalytic acid/base), respectively, of the known enzymes (S3 Fig). In fact, the D270A and E473A mutants showed no detectable hydrolytic activity toward pNP-β-Glc. This result supports the assignment of Asp270 and Glu473 as the catalytic nucleophile and catalytic acid/base residues, respectively. A distance between side chain carboxyl oxygen atoms of Asp270 and Glu473 is approximately 6.0 Å, suggesting that the enzyme follows retaining mechanism.

The Lin1840r-Glc complex structure was determined to understand the substrate recognition at subsite −1. Six residues (Asp91, Arg149, Lys191, His192, Asp270, and Glu473) constitute subsite −1 and form hydrogen bonds with the Glc molecule (S3 Fig). These residues can be well superimposed on the corresponding residues of the known structures of GH3 BGLs.

Complexes with inhibitors

To clarify the binding modes of inhibitors, Lin1840r-inhibitor complex structures were determined. In complex structures with IFG and GDL, which are Glc analogs, the ligands are located at the same position as Glc (Fig 2A, 2B and S3 Fig), suggesting that IFG and GDL compete with substrates for subsite −1. In the GDL complex, electron densities of glycerol molecules were observed between the aromatic rings of Tyr583 and Trp271 (Fig 2A). A GDL molecule can be fitted to a large electron density near Trp409 (outside the active center) and undergo hydrophobic stacking with Trp409, but the orientation of the molecule is obscure (Fig 2A). The structure of the GDL complex is not substantially different from those of the apo WT and Glc complex. In the case of the IFG complex structure, on the other hand, an unwound helix (amino acids 36–47) in domain 1 is inserted between the aromatic rings of Tyr583 and Trp271. Thr40 in this loop forms a hydrogen bond with the 6-OH group of IFG (Fig 2B). This loop might be involved in substrate binding in solution.

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Fig 2. Complexes with GDL (A), and IFG (B).

The two subunits comprising the catalytic pocket are shown in cyan and yellow. Residues involved in ligand binding are shown as sticks. The Leu39–Met47 loop seen in the IFG complex is shown in magenta and the disordered loop region (Gly32–Glu38) are shown in a black dotted line. Inhibitors and glycerol are represented as green and orange sticks, respectively. F_o−F_c electron density maps of GDL and IFG are shown as a gray mesh (contoured at 3.0σ and 4.0σ, respectively). GDL and glycerol (A) and IFG (B) were omitted for calculation of the F_o−F_c maps. Hydrogen bonds are presented as blue dotted lines.

https://doi.org/10.1371/journal.pone.0148870.g002

Complex structures with Sop₂ and Lam₂

In order to understand the recognition mechanism of Sop₂ and Lam₂, the complex structures with these disaccharides were determined using the nucleophile D270A mutant of Lin1840r. The electron density of Sop₂ was clearly observed in the D270A structure on soaking in 100 mM Sop₂ and fitted only with the β-anomer (Fig 3A left). The significant electron density of a ligand was also observed on soaking in 50 mM Lam₂ and clearly fitted with both anomers of Lam₂ (Fig 3B left). The Glc moieties of both Sop₂ and Lam₂ at subsite −1 are located at almost the same positions as the ligands in WT-inhibitors complexes despite the replacement of the catalytic nucleophile with alanine. The reducing end Glc moieties of Sop₂ and Lam₂ are consistently stacked on Trp271 and Tyr583, and form four hydrogen bonds with Arg572 (S3 Table), suggesting that these three residues constitute subsite +1. The fact that Tyr583 and Arg572 are derived from a loop in the adjacent subunit indicates the participation of both subunits in formation of the catalytic pocket.

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Fig 3. D270A-Sop₂ and D270A-Lam₂ complexes.

(A, B) Substrate pockets of D270A-Sop₂ (A) and D270A-Lam₂ (B) complexes. Ligands are shown as green sticks. Acid-base residues are shown in blue. (Left) Electron density maps of ligands. The F_o−F_c electron density maps of the ligands are presented as a gray mesh (contoured at 3.0σ). Sop₂ and Lam₂ are omitted for calculation of the F_o−F_c maps. Several hydroxy groups of ligands are labeled. Hydrogen bonds are presented as blue dotted lines. Lam₂ in the D270A-Lam₂ complex (B) is a mixture of both anomers. Distances between Arg572 and ligands are summarized in S3 Table. (Right) Conformations of ligands at subsite −1. The position of the catalytic nucleophile (Asp270) determined on superpositioning with apo WT is shown in red. The distances between two atoms linked by red dotted lines are given. (C) Comparison of conformation of Sop₂ with those of ligands in Michaelis complex and transition state-like complexes at subsite −1. Sop₂, Cel₂F-DNP in BaCel5A (Michaelis complex), and PheGlcIm in HvExoI (transition state) are shown in green, yellow, and cyan sticks, respectively. Catalytic residues of Lin1840r, HvExoI, and BaCel5A are shown in white, bright orange, and light blue, respectively.

https://doi.org/10.1371/journal.pone.0148870.g003

The results of Cremer-Pople ring pucker parameters calculator analysis (http://www.ric.hi-ho.ne.jp/asfushi/) [38] showed that the pyranose rings of Sop₂ and Lam₂ at subsite −1 are ⁴H₃ and ¹S₃, respectively. Since proposed itinerary of ring conformation to the substrate-enzyme intermediate in HvExoI is ⁴C₁ (pre-Michaelis complex) ⬄ ¹S₃ (Michaelis complex) ⬄ ⁴E (⁴H₃) (transition state) ⬄ ⁴C₁ (intermediate) [39,40], conformation of Lam₂ is considered to correspond with Michaelis complex. Sop₂ complex is also considered as Michaelis complex in spite of its conformation, since Sop₂ is not superimposed with glucophenylimidazole (PheGlcIm) known as a transition-like analog in HvExoI [40] but with ligands known as Michaelis complex, such as 2',4'-dinitrophenyl-2-deoxy-2-fluro-β-d-cellobioside (Cel₂F-DNP) in GH5 Bacillus agaradhaerens endo-β-1,4-glucanase (BaCel5A), a retaining enzyme [41] (Fig 3C). In addition, the Sop₂ molecule does not possess a trigonal anomeric center necessary to be judged as transition state. In the Sop₂ and Lam₂ complexes, the angles defined by the oxygen atom of the glycosidic bond, the anomeric carbon atoms in the ligands, and the carboxyl group in the catalytic nucleophile are 165° and 164°, respectively, as in the case of GlcNAc-MurNAc in a nucleophile mutant of GH3 B. subtillus N-acetyl-β-d-glucosaminidase and Cel₂F-DNP in BaCel5A [41,42]. Dihedral angles of Sop₂ and Lam₂ (O2-C1-O5-C5 in non-reducing end) are 93.4° and 88.3°, respectively, implying that the lone pairs on the both endocyclic oxygen atoms almost face antiperiplanar to the scissile bonds. These facts suggest that the nucleophile is able to mount an in-line attack on the anomeric carbon (Fig 3A and 3B right) [43]. The distances between the catalytic nucleophile and the anomeric carbon at subsite −1 are 3.2 Å and 3.0 Å, respectively, and the distances between the catalytic acid-base and oxygen atoms of the glycosidic bond are 3.4 Å and 3.2 Å, respectively (Fig 3A and 3B right). These distances are within the possible range of the reaction [44]. These facts suggest that the ligands are positioned, as they would be in catalytically active complexes. This is the first report of a Michaelis complex for GH3 BGLs, though pre-Michaelis complexes have been reported for HvExoI [39,40].

The anomeric hydroxy group of reducing end glucosides in Sop₂ is exposed to the solvent, in the vicinity of Asp91. In the case of Lam₂, the hydroxy group is exposed outside the substrate pocket, this being consistent with similar activity among Lam_ns.

Complexes with Sop₃, Cel₂, and Gen₂

Soaking of the D270A mutant crystal in 100 mM Sop₃ resulted in clear observation of electron density corresponding to the middle Glc moiety of Sop₃ at subsite +1 (S4A Fig). The electron densities of glycoside moieties at both the non-reducing and reducing ends might be derived from Sop₃ but they were so weak that interpretation was difficult. The electron density of the Glc moiety was not observed at subsite −1. In the Cel₂ and Gen₂ complex structures, electron densities of Glc moieties were also both observed at subsite +1 (S4B and S4C Fig). These Glc moieties were thought to be the non-reducing ends of Cel₂ and Gen₂ due to the orientation of the hydroxy group participating in the glycosidic linkage (S4B and S4C Fig), suggesting that Cel₂ or Gen₂ binds to the enzyme non-productively. In the both complexes, Glc molecules were observed only in molecule A. This might be due to impurity in quite high concentrations of soaked substrates. In the Cel₂ complex, Trp409 stacks on another Glc moiety unlike in the cases of the Sop₃ and Gen₂ complexes (S4 Fig).

Kinetic analysis of the R572K mutant

It is suggested that Arg572 is an important residue for the recognition of Sop₂, since it forms multiple hydrogen bonds with substrates at subsite +1 (Fig 3A and 3B left). We therefore characterized the R572K mutant. The kinetic parameters of the R572K mutant enzyme as to β-linked gluco-disaccharides are summarized in Table 1. The K_m value of the mutant enzyme for Sop₂ was 10 times higher than that of the WT enzyme. The mutant enzyme also showed increased K_m values for the other substrates, and 1.5–3.0 times smaller k_cat values for Sop₂, Lam₂, and Cel₂. As a result, the k_cat/K_m value of the R572K mutant for Sop₂ was over 25 times smaller, while the extent of reduction of the k_cat/K_m value for Lam₂ caused by the mutation was approximately 6 times. This indicates that Arg572 is important for substrate binding, especially for Sop₂.

Comparison of substrate pockets

In order to investigate the importance of Arg572 and subsite +2, the substrate pocket of Lin1840r was compared with those of JMB19063, a homolog in the same clade as Lin1840, and homologs in the different clade from Lin1840. Trp271, Tyr583, and Arg572 constitute subsite +1 in Lin1840r. They can be well superimposed on the corresponding residues in JMB19063 (Tyr262, Phe598, and Arg587, respectively) (Fig 4A). Try583 and Arg572 are derived from the other subunit, as observed for the corresponding residues of JMB19063. Contrarily, in the other structurally known GH3 BGLs, each subsite +1 comprises one subunit.

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Fig 4. Comparison of the active centers in Lin1840r and GH3 BGLs.

Apo WT Lin1840r is superimposed on (A) JMB19063, (B) HvExoI (PDB code 1IEX), (C) KmBglI (PDB code 3AC0), (D) AaBGL1 (PDB code 4IIG), (E) TrCel3A (PDB code 3ZYZ), and (F) TnBgl3B (PDB code 2X41). (Left) Domains 1 and 2 of apo WT Lin1840r are shown in cyan, and the loop extending from the other subunit is shown in yellow. The corresponding domains and loop of JMB19063 are shown in pink and green, respectively. HvExoI, KmBglI, AaBGL1, TrCel3A, and TnBgl3B are colored magenta, blue, brown, gray, and orange, respectively. Residues constituting the subsites are represented as sticks. The residue names in Lin1840r and the other enzymes are shown in red and black, respectively. (Right) The D270A-Sop₂ complex was used in place of the apo WT enzyme. D270A is shown as a surface representation. The superimposed GH3 BGLs are shown as sticks. Sop₂ in the D270A-Sop₂ complex is represented as a green stick. All ligands shown in the left figures are omitted in the right figures. (A) An anomeric hydroxy group of Sop₂ is labeled.

https://doi.org/10.1371/journal.pone.0148870.g004

The Trp271 residue is conserved in other structurally available GH3 BGLs, including HvExoI, β-glucosidase from Kluyveromyces marxianus (KmBglI) [45], AaBGL1 [33], β-glucosidase from Trichoderma reesei (TrCel3A) [46] which act on a variety of substrates containing cellobiose unlike Lin1840r, and aryl β-glucosidase from Thermotoga neapolitana (TnBgl3B) [47]. The corresponding residues in most GH3 BGLs are aromatic amino acids. The Trp286 residue in HvExoI stacks on the Glc moiety of the ligand at subsite +1, while Trp226 of KmBglI, Trp281 of AaBGL1, Trp237 of TrCel3A, and Trp243 of TnBgl3B participate in recognition of ligands at subsite −1 (Fig 4B–4F).

At the position corresponding to the Tyr583 residue, most structurally known GH3 BGLs have an aromatic amino acid, while the TnBgl3B structure lacks an aromatic residue probably due to disorder of the corresponding region. The Tyr583 residue and the corresponding residues of JMB19063, KmBglI, AaBGL1, and TrCel3A (Phe598, Phe508, Tyr511, and Trp443, respectively) are consistently located on loops extending from the acid-base catalyst residue side (Fig 4A and 4C–4E). On the other hand, Trp434 in HvExoI is on the loop extending from the opposite side (Fig 4B). Lin1840r and JMB19063 consistently lack this loop, and instead have empty spaces sufficiently large for glycoside binding (Fig 4A). This space is considered to be subsite +2, since the anomeric hydroxy group of reducing end glucosides in Sop₂ faces the space (Fig 4A right). The spaces are filled with loops in KmBglI, AaBGL1, and TrCel3A (Fig 4C–4E).

The Arg572 residue in the substrate pocket is located at a similar position to Arg291 of HvExoI (Fig 4B). However, Arg291 does not correspond with the Arg572 residue in terms of primary sequence, and HvExoI does not have any corresponding loop for the Arg572 residue. The Arg291 residue is localized farther away from the center of the catalytic pocket than Arg572 (Fig 4B). KmBglI, AaBGL1, TrCel3A, and TnBgl3B possess no residue that corresponds to Arg572 or Arg291 (Fig 4C–4F). Gly57 in the loop of HvExoI (amino acids 54–66) participates in substrate recognition on the opposite side of subsite +1 from Arg291 [48] (Fig 4B). KmBglI, AaBGL1, and TrCel3A possess Phe55, Arg98, and Arg67, respectively, at the position corresponding to Gly57 (Fig 4C–4E). On the other hand, Lin1840r has no corresponding loop (Fig 4A).

Discussion

In this study, the hydrolytic activity of Lin1840r toward Sop_ns was determined since the lin1839 gene in the same gene cluster as lin1840 encodes an enzyme specific to β-1,2-glucan. Lin1840r showed obvious preference for Sop₂ among Sop_ns (Table 1). Considering that Lin1839 phosphorolyzes β-1,2-glucan with DP 3 or more to produce G1P but does not act on Sop₂ [14], Lin1840 and Lin1839 cooperatively metabolize β-1,2-glucan in the cytosol. Since phosphorylation of glucose without the use of ATP is beneficial for energy acquisition, it is advantageous that BGLs show strong preference for substrates on which phosphorylases do not act. These facts suggest that Lin1840 is a BGL for Sop₂ degradation.

Nevertheless, structural analysis showed that Lin1840r possesses a sufficiently large space, which appears to be subsite +2, for access of Sop₃. This space is needed only for binding of Sop_ns, as judged from the orientations of the anomeric hydroxy groups of Sop₂ and Lam₂ in Lin1840r structures (Fig 3), and thioCel₂ (Fig 4B) [35] and methyl β-thiogentiobiose (PDB ID, 3WLP) in HvExoI structures. The corresponding spaces are filled in other structurally available GH3 BGLs except JMB19063 (Fig 4A–4E). However, the presence of Sop₃ accessible space at subsite +2 cannot explain the kinetic result that Lin1840r showed a much higher K_m value toward Sop₃ than that toward Sop₂. The conformation of Sop₃ is likely related to the difference in the K_m values. According to molecular dynamics simulation, Sop₃ adopts a stable conformation that includes an intramolecular hydrogen bond between the 3-OH group of the reducing end glucoside and the oxygen atom of the pyranose ring or 6-OH group of the non-reducing end glucoside [49]. These intramolecular hydrogen bonds in Sop₃ have to be removed and the glycosidic bond of Sop₃ at the reducing end also has to be distorted for productive binding of Sop₃. Overall, subsite +2 might be an evolutionary relic of specialization of Lin1840 from a Sop_ns degrading enzyme to one specialized at Sop₂ degradation.

In spite of the estimated function of Lin1840, Lin1840r shows comparable activity toward Lam₂ with Sop₂. The presence of Arg572 at subsite +1 and the space of subsite +2 are important features for substrate recognition. Arg572 forms many hydrogen bonds with Sop₂ or Lam₂ at subsite +1, and thereby likely compensates for the lack of any hydrogen-bond interaction with the substrates on the subsite +2 side (Figs 3A and 5A). The binding modes of Sop₂ and Lam₂ are apparently very similar in the Lin1840r complexes. The structures of the bound Sop₂ and Lam₂ molecules only differ in the substituting groups of reducing end glucosides on the subsite +2 side (Fig 5). This observation thus indicates that Lin1840r is not able to distinguish between Sop₂ and Lam₂. BGLs from C. arvensicola and Acremonium sp. 15 induced by β-1,2-glucan show similar substrate specificities for β-linked gluco-disaccharides to Lin1840 [12, 13]. They might have similar structural features to Lin1840r, though their amino acid sequences are unavailable.

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Fig 5. Comparison of conformations of ligands in Lin1840r and HvExoI.

Hydrogen bonds are represented as blue dotted lines. Subsites −1, +1, and +2 are labeled. (A) Comparison of Sop₂ and Lam₂ in the D270A mutant. Arg572 and Sop₂ in the D270A-Sop₂ complex are shown in cyan and green, respectively. Arg572 and Lam₂ of both anomers in the D270A-Lam₂ complex are colored orange and blue, respectively. Putative subsite +2 for Sop_ns and potential subsite +2 for Lam_ns are shown in a solid and dotted circle, respectively. An anomeric hydroxy group of a reducing-end glucoside in Sop₂ is labeled. (B) Comparison of Sop₂ in the D270A mutant and thioCel₂ in HvExoI. The residue and ligand in the D270A-Sop₂ complex are shown in the same as in (A). Arg291 and thioCel₂ in HvExoI-thioCel₂ are presented in gray and magenta, respectively.

https://doi.org/10.1371/journal.pone.0148870.g005

Unlike Lam₂, Arg572 effectively excludes Cel₂ as a substrate. This can be explained by comparison of the D270A-Sop₂ and HvExoI-thiocellobiose (thioCel₂) complex structures. Arg291 in HvExoI, which corresponds to Arg572 in Lin1840r, is located farther from the center of the substrate pocket than Arg572 (Figs 4B and 5B). The 6-OH group of the reducing end glucoside of thioCel₂ forms a hydrogen bond with Arg291. However, the distance between Arg572 and the 6-OH of the glucoside in thioCel₂ is so close as to cause steric hindrance (Fig 5B). Actually, Cel₂ binds to Lin1840r in the non-productive form. In addition, other structurally available GH3 BGLs (TnBgl3B, TrCel3A, AaBGL1 and KmBglI), which lack a residue corresponding to Arg572 (Fig 4C–4F), consistently show sufficient hydrolytic activity toward Cel₂ [45,46,47,50]. These observations suggest that Arg572 accounts for much higher K_m value for Cel₂ than those for Sop₂ and Lam₂. Considering that Sop₂ and Lam₂ complexes mimic Michaelis complex, Arg572 is also related with differences in k_cat values between Cel₂ and Sop₂/Lam₂. Thus, Arg572 is important for substrate specificity in Lin1840r.

The Arg572 residue is highly conserved among closely related homologs such as JMB19063 and FmBGL. Nevertheless, JMB19063 is thought to be involved in cellulose degradation and FmBGL is described as an aryl β-glucosidase. JMB19063 shares structural features important for substrate specificity with Lin1840. Therefore, we compared the substrate specificities of the three enzymes. The specific activity of JMB19063 toward Cel₂ (0.1 mM Cel₂ at 50°C) estimated from the substrate consumption is only 0.077 (U/mg), whereas JMB19063 shows high hydrolytic activity toward pNP-β-Glc (11.8 U/mg on 0.1 mM pNP-β-Glc) [18]. The specific activities of Lin1840r for pNP-β-Glc and Cel₂ (0.1 mM at 20°C) are 1.12 (U/mg) and 8.9 × 10⁻⁴ (U/mg), respectively. In addition, FmBGL shows similar levels of kinetic parameters for pNP-β-Glc (k_cat = 39.1 s⁻¹, K_m = 0.49 mM) to Lin1840r but at least 100 times lower activity toward Cel₂, as judged from preliminary results, than toward pNP-β-Glc [19,32]. Thus, the three enzymes show a similar tendency in substrate specificity as to pNP-β-Glc and Cel₂. It should be noted that Lin1840r shows comparable activity toward Sop₂ and Lam₂ as glycoside hydrolases, while the activities of JMB19063 and FmBGL toward Sop₂ and Lam₂ have not been reported. Moreover, the structure of the Lin1840r-Cel₂ complex was compared with that of the JMB19063-Cel₅ complex, which contains Glc in subsite −1 and Cel₂ outside subsite −1 [18] (Fig 4A). This complex is the same as the Cel₂ complex of Lin1840r in that subsites +1 and −1 are filled with separate molecules (S4B Fig). Moreover, while ThioCel₂ in the HvExoI-thioCel₂ complex forms the intrinsically stable conformation of Cel₂, glucoside at subsite +1 in the Cel₅-soaked JMB19063 complex is inverted compared to that of ThioCel₂ (Figs 4A and 5A). Thus, the Cel₅-soaked JMB19063 complex seems to be in the non-productive form, as in the case of the Cel₂-Lin1840r complex. Overall, it would not be surprising if JMB19063 and FmBGL show similar substrate specificity to Lin1840r.

This study strongly suggests that Lin1840 is a BGL for Sop₂ degradation, and that Arg572 and subsite +2 are the key factors for its substrate specificity. This is not only a significant revelation in the field of β-1,2-glucan metabolizing enzymes but also evoke the need for reevaluation of the functions of closely related homologs with Lin1840 possessing arginine residues corresponding with the Arg572.

Supporting Information

S1 Fig. Multiple alignment of closely related homologs with Lin1840.

Multiple alignment was performed using T-COFFEE multiple alignment server (http://tcoffee.vital-it.ch/apps/tcoffee/index.html) [51]. The amino acid sequences are selected evenly among the clade of Lin1840. The amino acid sequence of JMB19063 is based on the PDB (accession number, 3U48). The GenBank accession number of FmBGL is AAB66561.1. Uncharacterized proteins are presented as accession numbers. AEB30119.1, AEE54288.1, AEW02328.1, EEV33587.1, ELK47769.1, ERI92988.1, and ETT38232.1 are the GenBank accession numbers of homologous genes from Carnobacterium sp. 17–4, Haliscomenobacter hydrossis DSM 1100, Niastella koreensis GR20-10, Enterococcus gallinarum EG2, Halobacillus sp. BAB-2008, Clostridiales bacterium oral taxon 876, and Paenibacillus sp. FSL R5-808, respectively. 515950490 and 544876686 are the Geneinfo identifier numbers of homologous genes from Paenisporosarcina sp. TG-14 and Virgibacillus sp. CM-4, respectively. Conserved residues are indicated by asterisks. Arg572 in Lin1840 is indicated by an asterisk in inverted monochrome.

https://doi.org/10.1371/journal.pone.0148870.s001

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S2 Fig. Comparison of Lin1840r and JMB19063.

Domains 1, 2, and 3 of Lin1840r are presented in cyan, orange, and magenta, respectively. The corresponding domains of JMB19063 (PDB code, 3U48) are in light brown, blue, and brown, respectively. The linker region of Lin1840r (amino acids 340–359) is shown in black. The loop region (amino acids 543–604) in Lin1840r and the corresponding loop in JMB19063 are labeled and shown in red and green, respectively. Mg²⁺ and Ca²⁺, and water are shown as green, red, and light blue spheres, respectively. (A-B) Superpositioning of the dimers of both Lin1840r and JMB19063. (A) Both subunits B are presented in semitransparent. (B) Left monomers rotated by 90° in the direction of the Y-axis. The N-terminal and C-terminal regions of both subunits are denoted by N and C, respectively. (C) Metal ion binding sites of Lin1840r. Residues involved in binding of metal ions are labeled and represented as sticks. Residues forming inter-subunit hydrogen bonds are shown as sticks. Hydrogen bonds are depicted as blue dotted lines. F_o−F_c electron density maps of metal ions and coordinating atoms are shown as a gray mesh (contoured at 3.0σ).

https://doi.org/10.1371/journal.pone.0148870.s002

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S3 Fig. Superimpositioning of subsites −1 of GH3 BGLs.

All residues and ligands are shown as sticks. Lin1840r, JMB19063 (PDB ID, 3U48), HvExoI (PDB ID, 1IEQ), and KmBglI (PDB ID, 3AC0) are colored cyan, green, orange, and magenta, respectively. Glc molecules in JMB19063, HvExoI, and KmBglI are shown in blue, pink, and olive, respectively. The amino acid residues in Lin1840r are labeled.

https://doi.org/10.1371/journal.pone.0148870.s003

(TIF)

S4 Fig. Complex structures of D270A with Sop₃ (A), Cel₂ (B), and Gen₂ (C).

Residues constituting subsites are shown as sticks. The F_o−F_c electron density maps of the ligands are represented as a gray mesh (contoured at 3.0σ). Glc molecules were omitted for calculation of the F_o−F_c maps. The color usage is as in Fig 2. Glc molecules are fitted to electron densities and shown in green stick. Glc moieties of the ligands are positioned at subsite +1 (A), subsite −1 and +1, and vicinity of Trp409 (B), and subsite −1 and +1 (C), respectively. (B, C) Regions derived from molecule A and B are shown in cyan and yellow, respectively.

https://doi.org/10.1371/journal.pone.0148870.s004

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S1 Table. Data collection and refinement statistics.

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S2 Table. Comparison of overall structures of Lin1840r and two other GH3 enzymes.

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S3 Table. Distances between Arg572 and ligands in D270A-Sop₂ and -Lam₂ complexes.

https://doi.org/10.1371/journal.pone.0148870.s007

(TIF)

Acknowledgments

This work was performed with the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2012G108 and 2014G509).

Author Contributions

Conceived and designed the experiments: MN H. Taguchi. Performed the experiments: MN RY KA YT NS H. Toyoizumi. Analyzed the data: MN RY AM H. Taguchi. Contributed reagents/materials/analysis tools: MN H. Taguchi. Wrote the paper: MN RY AM HN MK H. Taguchi.

References

1. Hisamatsu M (1992) Cyclic (1→2)-β-d-glucans (cyclosophorans) produced by Agrobacterium and Rhizobium species. Carbohydr Res. 231: 137–146. pmid:1394310
- View Article
- PubMed/NCBI
- Google Scholar
2. Breedveld MW, Miller KJ (1994) Cyclic β-glucans of members of the family Rhizobiaceae. Microbiol Rev 58: 145–161. pmid:8078434
- View Article
- PubMed/NCBI
- Google Scholar
3. Hisamatsu M, Amemura A, Matsuo T, Matsuda H, Harada T (1982) Cyclic (1→2)-β-d-glucan and the octasaccharide repeating-unit of succinoglycan produced by Agrobacterium. J Gen Microbiol 128: 1873–1879.
- View Article
- Google Scholar
4. Briones G, Iñón de Iannino N, Roset M, Vigliocco A, Paulo PS, Ugalde RA (2001) Brucella abortus cyclic β-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells. Infect Immun 69: 4528–4535. pmid:11401996
- View Article
- PubMed/NCBI
- Google Scholar
5. Gay-Fraret J, Ardissone S, Kambara K, Broughton WJ, Deakin WJ, Le Quéré A (2012) Cyclic-β-glucans of Rhizobium (Sinorhizobium) sp. strain NGR234 are required for hypo-osmotic adaptation, motility, and efficient symbiosis with host plants. FEMS Microbiol Lett 333: 28–36. pmid:22583376
- View Article
- PubMed/NCBI
- Google Scholar
6. Rigano LA, Payette C, Brouillard G, Marano MR, Abramowicz L, Torres PS, et al. (2007) Bacterial cyclic β-(1,2)-glucan acts in systemic suppression of plant immune responses. Plant Cell 19: 2077–2089. pmid:17601826
- View Article
- PubMed/NCBI
- Google Scholar
7. Arellano-Reynoso B, Lapaque N, Salcedo S, Briones G, Ciocchini AE, Ugalde R, et al. (2005) Cyclic β-1,2-glucan is a Brucella virulence factor required for intracellular survival. Nat Immunol 6: 618–625. pmid:15880113
- View Article
- PubMed/NCBI
- Google Scholar
8. Bohin JP (2000) Osmoregulated periplasmic glucans in Proteobacteria. FEMS Microbiol Lett 186: 11–19. pmid:10779706
- View Article
- PubMed/NCBI
- Google Scholar
9. Talaga P, Fournet B, Bohin JP (1994) Periplasmic glucans of Pseudomonas syringae pv. syringae. J Bacteriol 176: 6538–6544. pmid:7961404
- View Article
- PubMed/NCBI
- Google Scholar
10. Miller KJ, Kennedy EP, Reinhold VN (1986) Osmotic adaptation by gram-negative bacteria: possible role for periplasmic oligosaccharides. Science 231: 48–51. pmid:3941890
- View Article
- PubMed/NCBI
- Google Scholar
11. Schmidt S, Zietz M, Schreiner M, Rohn S, Kroh LW, Krumbein A (2010) Identification of complex, naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry. Rapid Commun Mass Spectrom 24: 2009–2022. pmid:20552580
- View Article
- PubMed/NCBI
- Google Scholar
12. Kitahara S, Edogawa S (1987) Cyclic (1→2)-β-d-glucan-hydrolyzing enzymes from Acremonium sp. 15; purification and some properties of endo-(l→2)-β-d-glucanase and β-d-glucosidase. Agric Biol Chem 51: 2701–2708.
- View Article
- Google Scholar
13. Mendoza NS, Amemura A (1983) (1,2)-β-d-Glucan-hydrolyzing enzymes in Cytophaga arvensicola: partial purification an some properties of endo-(1,2)-β-glucanase and β-d-glucosidase specific for (1,2)-and (1,3)-linkages. J Ferment Technol 61: 473–481.
- View Article
- Google Scholar
14. Nakajima M, Toyoizumi H, Abe K, Nakai H, Taguchi H, Kitaoka M (2014) 1,2-β-Oligoglucan phosphorylase from Listeria innocua. PLoS One 9: e92353. pmid:24647662
- View Article
- PubMed/NCBI
- Google Scholar
15. Buchrieser C, Rusniok C, Kunst F, Cossart P, Glaser P, Consortium L (2003) Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity. FEMS Immunol Med Microbiol 35: 207–213. pmid:12648839
- View Article
- PubMed/NCBI
- Google Scholar
16. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37: D233–238. pmid:18838391
- View Article
- PubMed/NCBI
- Google Scholar
17. Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T (2012) Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta. Appl Microbiol Biotechnol 93: 1989–1998. pmid:21850431
- View Article
- PubMed/NCBI
- Google Scholar
18. McAndrew RP, Park JI, Heins RA, Reindl W, Friedland GD, D'haeseleer P, et al. (2013) From soil to structure, a novel dimeric β-glucosidase belonging to glycoside hydrolase family 3 isolated from compost using metagenomic analysis. J Biol Chem 288: 14985–14992. pmid:23580647
- View Article
- PubMed/NCBI
- Google Scholar
19. Li YK, Chu SH, Sung YH (1998) Purification, characterization and mechanistic study of β-glucosidase from Flavobacterium meningosepticum (ATCC 13253). J Chin Chem Soc 45: 603–610.
- View Article
- Google Scholar
20. Nakajima M, Yoshida R, Miyanaga A, Taguchi H (2014) Crystallization and preliminary X-ray diffraction analysis of Lin1840, a putative β-glucosidase from Listeria innocua. Acta Crystallogr F Struct Biol Commun 70: 1398–1401. pmid:25286948
- View Article
- PubMed/NCBI
- Google Scholar
21. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Prot Sci 4: 2411–2423.
- View Article
- Google Scholar
22. Abe K, Nakajima M, Kitaoka M, Toyoizumi H, Takahashi Y, Sugimoto N, et al. (2015) Large-scale preparation of 1,2-β-glucan using 1,2-β-oligoglucan phosphorylase. J Appl Glycosci 62: 47–52.
- View Article
- Google Scholar
23. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67: 271–281. pmid:21460445
- View Article
- PubMed/NCBI
- Google Scholar
24. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201. pmid:16301204
- View Article
- PubMed/NCBI
- Google Scholar
25. Collaborative Computational Project—Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763. pmid:15299374
- View Article
- PubMed/NCBI
- Google Scholar
26. Morris RJ, Perrakis A, Lamzin VS (2002) ARP/wARP's model-building algorithms. I. The main chain. Acta Crystallogr D Biol Crystallogr 58: 968–975. pmid:12037299
- View Article
- PubMed/NCBI
- Google Scholar
27. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132. pmid:15572765
- View Article
- PubMed/NCBI
- Google Scholar
28. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255. pmid:15299926
- View Article
- PubMed/NCBI
- Google Scholar
29. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372: 774–797. pmid:17681537
- View Article
- PubMed/NCBI
- Google Scholar
30. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786. pmid:21959131
- View Article
- PubMed/NCBI
- Google Scholar
31. Chir J, Withers S, Wan CF, Li YK (2002) Identification of the two essential groups in the family 3 β-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis. Biochem J 365: 857–863. pmid:11978178
- View Article
- PubMed/NCBI
- Google Scholar
32. Li YK, Chir J, Tanaka S, Chen FY (2002) Identification of the general acid/base catalyst of a family 3 β-glucosidase from Flavobacterium meningosepticum. Biochemistry 41: 2751–2759. pmid:11851422
- View Article
- PubMed/NCBI
- Google Scholar
33. Suzuki K, Sumitani J, Nam YW, Nishimaki T, Tani S, Wakagi T, et al. (2013) Crystal structures of glycoside hydrolase family 3 β-glucosidase 1 from Aspergillus aculeatus. Biochem J 452: 211–221. pmid:23537284
- View Article
- PubMed/NCBI
- Google Scholar
34. Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38: W545–549. pmid:20457744
- View Article
- PubMed/NCBI
- Google Scholar
35. Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7: 179–190. pmid:10368285
- View Article
- PubMed/NCBI
- Google Scholar
36. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. (2014) Pfam: the protein families database. Nucleic Acids Res 42: D222–230. pmid:24288371
- View Article
- PubMed/NCBI
- Google Scholar
37. Zheng H, Chordia MD, Cooper DR, Chruszcz M, Müller P, Sheldrick GM, et al. (2014) Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server. Nat Protoc 9: 156–170. pmid:24356774
- View Article
- PubMed/NCBI
- Google Scholar
38. Cremer D, Pople JA (1975) A general definition of ring puckering coordinates. J Am Chem Soc 97: 1354–1358.
- View Article
- Google Scholar
39. Hrmova M, Varghese JN, De Gori R, Smith BJ, Driguez H, Fincher GB (2001) Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant β-d-glucan glucohydrolase. Structure 9: 1005–1016. pmid:11709165
- View Article
- PubMed/NCBI
- Google Scholar
40. Hrmova M, De Gori R, Smith BJ, Vasella A, Varghese JN, Fincher GB (2004) Three-dimensional structure of the barley β-d-glucan glucohydrolase in complex with a transition state mimic. J Biol Chem 279: 4970–4980. pmid:14597633
- View Article
- PubMed/NCBI
- Google Scholar
41. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, Schülein M, et al. (1998) Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry 37: 11707–13. pmid:9718293
- View Article
- PubMed/NCBI
- Google Scholar
42. Bacik JP, Whitworth GE, Stubbs KA, Vocadlo DJ, Mark BL (2012) Active site plasticity within the glycoside hydrolase NagZ underlies a dynamic mechanism of substrate distortion. Chem Biol 19: 1471–1482. pmid:23177201
- View Article
- PubMed/NCBI
- Google Scholar
43. Vasella A, Davies GJ, Böhm M (2002) Glycosidase mechanisms. Curr Opin Chem Biol 6: 619–629. pmid:12413546
- View Article
- PubMed/NCBI
- Google Scholar
44. Hovel K, Shallom D, Niefind K, Belakhov V, Shoham G, Baasov T, et al. (2003) Crystal structure and snapshots along the reaction pathway of a family 51 α-l-arabinofuranosidase. EMBO J 22: 4922–4932. pmid:14517232
- View Article
- PubMed/NCBI
- Google Scholar
45. Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, et al. (2010) Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus. Biochem J 431: 39–49. pmid:20662765
- View Article
- PubMed/NCBI
- Google Scholar
46. Karkehabadi S, Helmich KE, Kaper T, Hansson H, Mikkelsen NE, Gudmundsson M, et al. (2014) Biochemical characterization and crystal structures of a fungal family 3 β-glucosidase, Cel3A from Hypocrea jecorina. J Biol Chem 289: 31624–31637. pmid:25164811
- View Article
- PubMed/NCBI
- Google Scholar
47. Pozzo T, Pasten JL, Karlsson EN, Logan DT (2010) Structural and functional analyses of β-glucosidase 3B from Thermotoga neapolitana: a thermostable three-domain representative of glycoside hydrolase 3. J Mol Biol 397: 724–739. pmid:20138890
- View Article
- PubMed/NCBI
- Google Scholar
48. Hrmova M, De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, et al. (2002) Structural basis for broad substrate specificity in higher plant β-d-glucan glucohydrolases. Plant Cell 14: 1033–1052. pmid:12034895
- View Article
- PubMed/NCBI
- Google Scholar
49. Stortz CA, Cerezo AS (2003) MM3 potential energy surfaces of the 2-linked glucosyl trisaccharides α-kojitriose and β-sophorotriose. Carbohydr Res 338: 1679–1689. pmid:12873423
- View Article
- PubMed/NCBI
- Google Scholar
50. Baba Y, Sumitani JI, Tani S, Kawaguchi T (2015) Characterization of Aspergillus aculeatus β-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system. AMB Express 5: 3. pmid:25642400
- View Article
- PubMed/NCBI
- Google Scholar
51. Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang JM, et al. (2007) T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res 39: W13–17.
- View Article
- Google Scholar

[ref1] 1. Hisamatsu M (1992) Cyclic (1→2)-β-d-glucans (cyclosophorans) produced by Agrobacterium and Rhizobium species. Carbohydr Res. 231: 137–146. pmid:1394310
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Breedveld MW, Miller KJ (1994) Cyclic β-glucans of members of the family Rhizobiaceae. Microbiol Rev 58: 145–161. pmid:8078434
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Hisamatsu M, Amemura A, Matsuo T, Matsuda H, Harada T (1982) Cyclic (1→2)-β-d-glucan and the octasaccharide repeating-unit of succinoglycan produced by Agrobacterium. J Gen Microbiol 128: 1873–1879.
View Article
Google Scholar

[10] View Article

[11] Google Scholar

[ref4] 4. Briones G, Iñón de Iannino N, Roset M, Vigliocco A, Paulo PS, Ugalde RA (2001) Brucella abortus cyclic β-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells. Infect Immun 69: 4528–4535. pmid:11401996
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Gay-Fraret J, Ardissone S, Kambara K, Broughton WJ, Deakin WJ, Le Quéré A (2012) Cyclic-β-glucans of Rhizobium (Sinorhizobium) sp. strain NGR234 are required for hypo-osmotic adaptation, motility, and efficient symbiosis with host plants. FEMS Microbiol Lett 333: 28–36. pmid:22583376
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Rigano LA, Payette C, Brouillard G, Marano MR, Abramowicz L, Torres PS, et al. (2007) Bacterial cyclic β-(1,2)-glucan acts in systemic suppression of plant immune responses. Plant Cell 19: 2077–2089. pmid:17601826
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Arellano-Reynoso B, Lapaque N, Salcedo S, Briones G, Ciocchini AE, Ugalde R, et al. (2005) Cyclic β-1,2-glucan is a Brucella virulence factor required for intracellular survival. Nat Immunol 6: 618–625. pmid:15880113
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Bohin JP (2000) Osmoregulated periplasmic glucans in Proteobacteria. FEMS Microbiol Lett 186: 11–19. pmid:10779706
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Talaga P, Fournet B, Bohin JP (1994) Periplasmic glucans of Pseudomonas syringae pv. syringae. J Bacteriol 176: 6538–6544. pmid:7961404
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Miller KJ, Kennedy EP, Reinhold VN (1986) Osmotic adaptation by gram-negative bacteria: possible role for periplasmic oligosaccharides. Science 231: 48–51. pmid:3941890
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref11] 11. Schmidt S, Zietz M, Schreiner M, Rohn S, Kroh LW, Krumbein A (2010) Identification of complex, naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry. Rapid Commun Mass Spectrom 24: 2009–2022. pmid:20552580
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref12] 12. Kitahara S, Edogawa S (1987) Cyclic (1→2)-β-d-glucan-hydrolyzing enzymes from Acremonium sp. 15; purification and some properties of endo-(l→2)-β-d-glucanase and β-d-glucosidase. Agric Biol Chem 51: 2701–2708.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref13] 13. Mendoza NS, Amemura A (1983) (1,2)-β-d-Glucan-hydrolyzing enzymes in Cytophaga arvensicola: partial purification an some properties of endo-(1,2)-β-glucanase and β-d-glucosidase specific for (1,2)-and (1,3)-linkages. J Ferment Technol 61: 473–481.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref14] 14. Nakajima M, Toyoizumi H, Abe K, Nakai H, Taguchi H, Kitaoka M (2014) 1,2-β-Oligoglucan phosphorylase from Listeria innocua. PLoS One 9: e92353. pmid:24647662
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Buchrieser C, Rusniok C, Kunst F, Cossart P, Glaser P, Consortium L (2003) Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity. FEMS Immunol Med Microbiol 35: 207–213. pmid:12648839
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37: D233–238. pmid:18838391
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T (2012) Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta. Appl Microbiol Biotechnol 93: 1989–1998. pmid:21850431
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. McAndrew RP, Park JI, Heins RA, Reindl W, Friedland GD, D'haeseleer P, et al. (2013) From soil to structure, a novel dimeric β-glucosidase belonging to glycoside hydrolase family 3 isolated from compost using metagenomic analysis. J Biol Chem 288: 14985–14992. pmid:23580647
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref19] 19. Li YK, Chu SH, Sung YH (1998) Purification, characterization and mechanistic study of β-glucosidase from Flavobacterium meningosepticum (ATCC 13253). J Chin Chem Soc 45: 603–610.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref20] 20. Nakajima M, Yoshida R, Miyanaga A, Taguchi H (2014) Crystallization and preliminary X-ray diffraction analysis of Lin1840, a putative β-glucosidase from Listeria innocua. Acta Crystallogr F Struct Biol Commun 70: 1398–1401. pmid:25286948
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref21] 21. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Prot Sci 4: 2411–2423.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref22] 22. Abe K, Nakajima M, Kitaoka M, Toyoizumi H, Takahashi Y, Sugimoto N, et al. (2015) Large-scale preparation of 1,2-β-glucan using 1,2-β-oligoglucan phosphorylase. J Appl Glycosci 62: 47–52.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref23] 23. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67: 271–281. pmid:21460445
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref24] 24. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201. pmid:16301204
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref25] 25. Collaborative Computational Project—Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763. pmid:15299374
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref26] 26. Morris RJ, Perrakis A, Lamzin VS (2002) ARP/wARP's model-building algorithms. I. The main chain. Acta Crystallogr D Biol Crystallogr 58: 968–975. pmid:12037299
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref27] 27. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132. pmid:15572765
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref28] 28. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255. pmid:15299926
View Article
PubMed/NCBI
Google Scholar

[104] View Article

[105] PubMed/NCBI

[106] Google Scholar

[ref29] 29. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372: 774–797. pmid:17681537
View Article
PubMed/NCBI
Google Scholar

[108] View Article

[109] PubMed/NCBI

[110] Google Scholar

[ref30] 30. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786. pmid:21959131
View Article
PubMed/NCBI
Google Scholar

[112] View Article

[113] PubMed/NCBI

[114] Google Scholar

[ref31] 31. Chir J, Withers S, Wan CF, Li YK (2002) Identification of the two essential groups in the family 3 β-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis. Biochem J 365: 857–863. pmid:11978178
View Article
PubMed/NCBI
Google Scholar

[116] View Article

[117] PubMed/NCBI

[118] Google Scholar

[ref32] 32. Li YK, Chir J, Tanaka S, Chen FY (2002) Identification of the general acid/base catalyst of a family 3 β-glucosidase from Flavobacterium meningosepticum. Biochemistry 41: 2751–2759. pmid:11851422
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref33] 33. Suzuki K, Sumitani J, Nam YW, Nishimaki T, Tani S, Wakagi T, et al. (2013) Crystal structures of glycoside hydrolase family 3 β-glucosidase 1 from Aspergillus aculeatus. Biochem J 452: 211–221. pmid:23537284
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref34] 34. Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38: W545–549. pmid:20457744
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref35] 35. Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7: 179–190. pmid:10368285
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref36] 36. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. (2014) Pfam: the protein families database. Nucleic Acids Res 42: D222–230. pmid:24288371
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref37] 37. Zheng H, Chordia MD, Cooper DR, Chruszcz M, Müller P, Sheldrick GM, et al. (2014) Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server. Nat Protoc 9: 156–170. pmid:24356774
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref38] 38. Cremer D, Pople JA (1975) A general definition of ring puckering coordinates. J Am Chem Soc 97: 1354–1358.
View Article
Google Scholar

[144] View Article

[145] Google Scholar

[ref39] 39. Hrmova M, Varghese JN, De Gori R, Smith BJ, Driguez H, Fincher GB (2001) Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant β-d-glucan glucohydrolase. Structure 9: 1005–1016. pmid:11709165
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref40] 40. Hrmova M, De Gori R, Smith BJ, Vasella A, Varghese JN, Fincher GB (2004) Three-dimensional structure of the barley β-d-glucan glucohydrolase in complex with a transition state mimic. J Biol Chem 279: 4970–4980. pmid:14597633
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref41] 41. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, Schülein M, et al. (1998) Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry 37: 11707–13. pmid:9718293
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref42] 42. Bacik JP, Whitworth GE, Stubbs KA, Vocadlo DJ, Mark BL (2012) Active site plasticity within the glycoside hydrolase NagZ underlies a dynamic mechanism of substrate distortion. Chem Biol 19: 1471–1482. pmid:23177201
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref43] 43. Vasella A, Davies GJ, Böhm M (2002) Glycosidase mechanisms. Curr Opin Chem Biol 6: 619–629. pmid:12413546
View Article
PubMed/NCBI
Google Scholar

[163] View Article

[164] PubMed/NCBI

[165] Google Scholar

[ref44] 44. Hovel K, Shallom D, Niefind K, Belakhov V, Shoham G, Baasov T, et al. (2003) Crystal structure and snapshots along the reaction pathway of a family 51 α-l-arabinofuranosidase. EMBO J 22: 4922–4932. pmid:14517232
View Article
PubMed/NCBI
Google Scholar

[167] View Article

[168] PubMed/NCBI

[169] Google Scholar

[ref45] 45. Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, et al. (2010) Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus. Biochem J 431: 39–49. pmid:20662765
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref46] 46. Karkehabadi S, Helmich KE, Kaper T, Hansson H, Mikkelsen NE, Gudmundsson M, et al. (2014) Biochemical characterization and crystal structures of a fungal family 3 β-glucosidase, Cel3A from Hypocrea jecorina. J Biol Chem 289: 31624–31637. pmid:25164811
View Article
PubMed/NCBI
Google Scholar

[175] View Article

[176] PubMed/NCBI

[177] Google Scholar

[ref47] 47. Pozzo T, Pasten JL, Karlsson EN, Logan DT (2010) Structural and functional analyses of β-glucosidase 3B from Thermotoga neapolitana: a thermostable three-domain representative of glycoside hydrolase 3. J Mol Biol 397: 724–739. pmid:20138890
View Article
PubMed/NCBI
Google Scholar

[179] View Article

[180] PubMed/NCBI

[181] Google Scholar

[ref48] 48. Hrmova M, De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, et al. (2002) Structural basis for broad substrate specificity in higher plant β-d-glucan glucohydrolases. Plant Cell 14: 1033–1052. pmid:12034895
View Article
PubMed/NCBI
Google Scholar

[183] View Article

[184] PubMed/NCBI

[185] Google Scholar

[ref49] 49. Stortz CA, Cerezo AS (2003) MM3 potential energy surfaces of the 2-linked glucosyl trisaccharides α-kojitriose and β-sophorotriose. Carbohydr Res 338: 1679–1689. pmid:12873423
View Article
PubMed/NCBI
Google Scholar

[187] View Article

[188] PubMed/NCBI

[189] Google Scholar

[ref50] 50. Baba Y, Sumitani JI, Tani S, Kawaguchi T (2015) Characterization of Aspergillus aculeatus β-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system. AMB Express 5: 3. pmid:25642400
View Article
PubMed/NCBI
Google Scholar

[191] View Article

[192] PubMed/NCBI

[193] Google Scholar

[ref51] 51. Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang JM, et al. (2007) T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res 39: W13–17.
View Article
Google Scholar

[195] View Article

[196] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Preparation of recombinant Lin1840 and mutants

Enzyme assays using p-nitrophenyl-sugars

Kinetic analysis using pNP-β-Glc and β-linked gluco-oligosaccharides

Temperature and pH profiles

Inhibition kinetics

Crystallography

Results

General properties of Lin1840r

Substrate specificity

Inhibition kinetics

Overall structure

Subsite −1 of Lin1840r

Complexes with inhibitors

Complex structures with Sop2 and Lam2

Complexes with Sop3, Cel2, and Gen2

Kinetic analysis of the R572K mutant

Comparison of substrate pockets

Discussion

Supporting Information

S1 Fig. Multiple alignment of closely related homologs with Lin1840.

S2 Fig. Comparison of Lin1840r and JMB19063.

S3 Fig. Superimpositioning of subsites −1 of GH3 BGLs.

S4 Fig. Complex structures of D270A with Sop3 (A), Cel2 (B), and Gen2 (C).

S1 Table. Data collection and refinement statistics.

S2 Table. Comparison of overall structures of Lin1840r and two other GH3 enzymes.

S3 Table. Distances between Arg572 and ligands in D270A-Sop2 and -Lam2 complexes.

Acknowledgments

Author Contributions

References

Complex structures with Sop₂ and Lam₂

Complexes with Sop₃, Cel₂, and Gen₂

S4 Fig. Complex structures of D270A with Sop₃ (A), Cel₂ (B), and Gen₂ (C).

S3 Table. Distances between Arg572 and ligands in D270A-Sop₂ and -Lam₂ complexes.