Article ID: 7202104
Glycoside hydrolase family 131 (GH131) proteins are found in oomycetes, ascomycetes, and basidiomycetes, and have been reported to hydrolyze various β-glucan polysaccharides. Coprinopsis cinerea, a model basidiomycete, contains two GH131 proteins, CcGH131A and CcGH131B. This study focuses on the structural and functional properties of CcGH131B, a protein that lacks the carbohydrate bonding module 1 (CBM1) domain present in CcGH131A. The crystal structure of CcGH131B was determined. The structure displayed a β-jelly roll fold with extra loops and α-helices, resulting in a deeper substrate-binding groove compared to CcGH131A and also PaGluc131A, a GH131 protein from Podospora anserina. A cellobiose-bound structure of the E161A mutant, in which the potential catalytic residue Glu161 was substituted with Ala, showed that the region of the minus subsites bind cellulose. In contrast, the region of the plus subsites mainly consists of hydrophobic amino acid residues and appeared to interact with hydrophobic molecules rather than with carbohydrates. Analysis using native affinity polyacrylamide gel electrophoresis showed that CcGH131B interacted with cellulosic polysaccharides such as methylcellulose and carboxymethylcellulose, while the protein exhibited no detectable enzymatic activity under the tested conditions. These results suggest that the substrate specificity of CcGH131B is likely to be different from those of CcGH131A and PaGluc131A.
CBM, carbohydrate bonding module; CcGH131A, Coprinopsis cinerea GH131A protein; CcGH131B, Coprinopsis cinerea GH131B protein; Cip1, cellulose induced protein 1; GH, glycoside hydrolase family; PaGluc131A, Podospora anserina Gluc131A protein; PL, polysaccharide lyase family.
Glycoside hydrolase family (GH) 131 is a small group of proteins in the Carbohydrate-Active enZymes (CAZy) database [1], with organisms found in cellulosic biomass-degrading oomycetes, ascomycetes, and basidiomycetes. The enzymatic properties of a GH131 protein from Podospora anserina, PaGluc131A, were first described in the previous studies [2, 3]. PaGluc131A has been identified as an unusual hydrolase that acts on various β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichnan, pustulan, and cellulosic derivatives. Several other GH131 proteins have also been reported to exhibit similar substrate specificities [4].
Coprinopsis cinerea is a basidiomycete that is recognized as a model mushroom-forming organism, and the whole genome sequence data is available [5]. In the previous paper, we focused on a GH131 protein, CcGH131A, encoded by a CC1G_07166 gene from C. cinerea [6]. CcGH131A shares 42 % sequence identity with PaGluc131A, and consists of an N-terminal catalytic domain and a carbohydrate binding module 1 (CBM1) domain as observed in PaGluc131A. We have determined the crystal structure of the catalytic domain of CcGH131A, which is composed of a β-jelly roll fold, with a wide and shallow substrate-binding groove. The crystal structure of the catalytic domain of PaGluc131A has also been determined [3], revealing an architecture essentially identical to that of CcGH131A.
It is intriguing that the biomass-degrading fungi often possess multiple GH131 proteins. Coprinopsis. cinerea possesses two GH131 proteins, CC1G_07166 and CC1G_15039, which we designated as CcGH131A and CcGH131B, respectively. The amino acid sequence identity between CcGH131B and CcGH131A is 30 %, which is lower than that between CcGH131A and PaGluc131A. Notably, CcGH131B lacks the CBM1 domain present in CcGH131A and PaGluc131A, suggesting that CcGH131B may play a distinct role in biomass degradation. Here we investigated the crystal structure and some properties of CcGH131B.
Construction of the expression plasmid for wild-type CcGH131B and the E161A mutant
The cDNA of C. cinerea was obtained as described [6]. The N-terminal signal peptide, predicted to comprise the 17 amino acid sequence MILSSLLWLCCYLGLVN, was identified using the SignalP server [7]. This sequence was omitted in the primer design. The primers, 5'-GGC AGC CAT ATG GGC AAG GTG TTG TGG GAT GGT AGG GCA-3', and 5'-AG TGC GGC GGC CGC AAC CTC CTG GAT GAA CTT GTT TCC-3' (the restriction sites for NdeI and NotI are underlined) were used for polymerase chain reaction amplification. The amplified fragment was ligated into the pET21a(+) vector (Merck Millipore Corporation, Burlington, MA, USA). The resultant recombinant protein was designed to have a His-tag (AAALEHHHHHH) at the C-terminus. The amplified sequence was found to be almost identical to the predicted exon sequence of CcGH131B in the database, but the two amino acid residues and their codons of CcGH131B used in this study were Ala87 (GCC) and Glu247 (GAG), while those of the corresponding residues and codons in the database were Thr87 (ACC) and Lys247 (AAG). The 87th and 247th residues are located far from the conserved Glu161 residue, and do not appear to be involved in the catalytic reaction. All experiments in this study were carried out using CcGH131B where the 87th and 247th residues are Ala and Glu, respectively, and the protein is referred to simply as CcGH131B in this paper. Site-directed mutagenesis to introduce the E161A mutation was performed using the QuikChange method [8] with the plasmid encoding wild-type CcGH131B as the template. A pair of oligonucleotides, 5'-C CAG CTT GTG TTT GTG GCG CCG TCC GAT GG-3' and 5'-G ACT CCC ATC GGA CGG CGC CAC AAA CAC AA-3', was used to construct the CcGH131B E161A mutant. The constructs were verified by DNA sequencing.
Crystallization, diffraction data collection, and structure determination
Wild-type CcGH131B and the E161A mutant were expressed in Escherichia coli BL21(DE3) and purified using the same procedure as described for CcGH131A [6]. The purified proteins were concentrated to 10 mg/mL in 10 mM sodium phosphate buffer (pH 7.5) using an Amicon Ultra-15 centrifugal unit (Merck Millipore Corporation). The concentration of the purified proteins was determined by measuring absorbance at 280 nm, using an extinction coefficient (1 mg/mL = 1.55) calculated by the Expasy ProtParam server (http://web.expasy.org/protparam/). The proteins were crystallized at 20 °C using the hanging drop vapor diffusion method. For wild-type CcGH131B, 1 μL of protein solution was mixed with an equal volume of well solution containing 3 % polyethylene glycol 10,000, 10 % polyethylene glycol 20,000, and 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH buffer (pH 6.5). The harvested crystal was cryoprotected with 40 % polyethylene glycol 20,000 and 0.1 M MES-NaOH buffer (pH 6.5). For the E161A mutant, 1 μL protein was mixed with the same volume of well solution containing 8 % polyethylene glycol 8,000, 0.1 M ammonium phosphate, and 0.1 M MES-NaOH buffer (pH 6.5). To obtain the complex structure of E161A-cellobiose, the harvested crystal was soaked in 20 % polyethylene glycol 8,000, 0.25 M cellobiose, 0.1 M ammonium phosphate, and 0.1 M MES-NaOH buffer (pH 6.5). To avoid the binding of common cryoprotectant molecules such as glycerol and ethylene glycol to the substrate-binding groove, high concentration of cellobiose was used for the soaking solution, and cellobiose also worked as the cryoprotectant. The crystals were flash-cooled in liquid nitrogen. Diffraction data were collected at beamlines AR-NE3A and AR-NW12A (Photon Factory, Tsukuba, Japan). Diffraction data images were processed using iMosflm [9] and scaled with AIMLESS [10] in the CCP4 program suite [11]. The structures were solved by the molecular replacement method with MOLREP [12] in CCP4 and a model of CcGH131A (PDB ID, 3W9A) [6] was employed as a probe model. Refinement was performed using REFMAC5 [13] in the CCP4 suite, and manual adjustment and rebuilding of the model were carried out using COOT [14]. The Ramachandran plot was calculated with the structure validation program RAMPAGE [15] in the CCP4 suite. The figures were produced using PyMOL (http://www.pymol.org/). The data collection and refinement statistics are summarized in Table 1. The coordinates and structure factors were deposited in the Protein Data Bank under the accession codes 9K7O and 9K7M.
Table 1. Data collection and refinement statistics.
| Unliganded CcGH131B | E161A-cellobiose | |
| Data collection | ||
| Beamline | KEK PF-AR NE3A | KEK PF-AR NW12A |
| Wavelength (Å) | 1.0 | 1.0 |
| Space group | P61 | P61 |
| Cell dimensions | ||
| a = b (Å) | 71.8 | 71.7 |
| c (Å) | 225.8 | 226.5 |
| Resolution range (Å) | 47.9-1.90 (1.97-1.90) | 48.0-1.45 (1.53-1.45) |
| Measured reflections | 485,997 | 1,436,242 |
| Unique reflections | 51,549 (5,122) | 115,588 (16,824) |
| Redundancy | 9.4 (9.3) | 12.4 (12.3) |
| Completeness (%) | 100 (100) | 99.7 (99.1) |
| <I/σ(I)> | 28.6 (4.0) | 16.9 (4.7) |
| Rmerge | 0.119 (0.457) | 0.087 (0.497) |
| Rp.i.m. | 0.041 (0.159) | 0.026 (0.146) |
| Refinement | ||
| Rwork | 0.180 | 0.162 |
| Rfree | 0.216 | 0.185 |
| Root mean square deviation (rmsd) | ||
| Bond lengths (Å) | 0.006 | 0.006 |
| Bond angles (°) | 1.391 | 1.380 |
| Ramachandran plot (RAMPAGE) | ||
| Favored (%) | 98.0 | 98.1 |
| Allowed (%) | 2.0 | 1.7 |
| Outliers (%) | 0 | 0.2 |
| Number of atoms | ||
| Protein | 4,793 | 4,790 |
| Water | 458 | 911 |
| Polyethylene glycol | 14 | 17 |
| Cellobiose | - | 46 |
| MES | - | 24 |
| Average B (Å2) | ||
| Protein | 30.3 | 17.0 |
| Water | 33.7 | 29.3 |
| Polyethylene glycol | 51.9 | 47.1 |
| Cellobiose | - | 13.6 |
| MES | - | 15.9 |
| PDB ID | 9K7O | 9K7M |
Values for the highest resolution shells are listed in parentheses.
-, Not applicable.
Characterization of the properties of CcGH131B
The following soluble carbohydrates were used to characterize the properties of CcGH131B: methylcellulose Metolose SM-15 and Metolose SM-100 (Shin-Etsu Chemical Co. Ltd., Tokyo, Japan), carboxymethylcellulose (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), birchwood xylan (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), soluble starch (Fujifilm Wako Pure Chemical Corporation), and laminarin (Nacalai Tesque, Inc., Kyoto, Japan). The insoluble carbohydrate, Avicel PH101 (Sigma-Aldrich Co. LLC) was also tested to evaluate whether CcGH131B showed enzymatic activity. Interaction of CcGH131B with soluble polysaccharides was assessed using native affinity polyacrylamide gel electrophoresis (PAGE) as described [16]. To test whether CcGH131B could degrade polysaccharides, CcGH131B (0.2 mg/mL) was incubated with 1 % (w/v) each polysaccharide in 10 mM sodium phosphate buffer (pH 6.5) at 37 °C for 20 h, and the mixtures were analyzed by thin-layer chromatography (TLC). For the insoluble Avicel PH101, CcGH131B (0.2 mg/mL) was incubated with the 1 % (w/v) suspension in 10 mM sodium phosphate buffer (pH 6.5) with vertical rotation at 12 rpm. Samples (1.0 μL) were taken and spotted on TLC plates of Silica Gel 60 (Merck Millipore Corporation) and developed with a mixture of 1-butanol:ethanol:water (5:5:2). The plates were dried, sprayed with 5 % (v/v) sulfuric acid-methanol solution, and charred at 110 °C.
To test whether CcGH131B could act on p-nitrophenyl β-D-cellobioside, 50 μL CcGH131B (1 mg/mL) and 50 μL p-nitrophenyl β-D-cellobioside (10 mM), both of which were in 10 mM sodium phosphate buffer (pH 6.0), were mixed and incubated at 37 °C. After incubation for 3, 6, or 9 h, 200 μL of alkaline solution consisting of 0.2 M Na2HPO4-NaOH (pH 11.5) was added to each tube, and the absorbance at 400 nm was measured.
Overall structure of CcGH131B
The crystal structure of unliganded CcGH131B was determined. The crystal contained two molecules, Mol-A and Mol-B, in the asymmetric unit, which are essentially identical. Structural analysis using the PISA server [17] indicated that no specific interaction between Mol-A and Mol-B was found, suggesting that CcGH131B exists as a monomer in solution. A Ramachandran plot was calculated with RAMPAGE, indicating that no residues in unliganded CcGH131B were outliers, whereas one residue, Pro78 in Mol-B of the E161A mutant (described later), was an outlier (Table 1). The electron density showed continuous density for most of the main chain atoms, but the 2|Fo|−|Fc| maps for residues 77-79 of both Mol-A and Mol-B were better resolved at the lower contour level of 0.8 σ. A portion of polyethylene glycol molecule was found in the same position on the molecular surface near Ser95 of both Mol-A and Mol-B.
The structure of CcGH131B consists of a β-jelly roll fold (Fig. 1A) as observed in other GH131 proteins. A structural similarity search was carried out using the DALI server (Table 2) [18]. Apart from homology with the GH131 proteins, CcGH131B is structurally similar to polysaccharide lyase family (PL) 20 [19], PL13 [20], and PL7 [21], as well as GH16 [22]. Additionally, CcGH131B showed homology with a domain in Clostridium botulinum ganglioside-binding protein [23], a domain in Bacteroides thetaiotaomicron GH143 enzyme [24], and uncharacterized protein Cip1 from Trichoderma reesei (Hypocrea jecorina) [25]. A comparison between the structures of CcGH131B and CcGH131A indicated that both proteins share a common structural framework (Fig. 1B). A topology diagram was generated using the PDBsum server [26] (Fig. 2). CcGH131B mainly consists of two β-sheets, Sheet-A and Sheet-B, both of which are also found in CcGH131A. Based on the numbering scheme for CcGH131A, β-strands in Sheet-A and Sheet-B are numbered A1-A9 and B1-B7, respectively (Fig. 2). In enzymes with a β-jelly roll fold, the substrate-binding sites are located in a groove formed by Sheet-B. CcGH131B contains five α-helices, H1-H5, with α-helices H1 and H2 conserved in CcGH131A.
(A) Stereo view of a ribbon model of CcGH131B. The common structures shared between CcGH131A and CcGH131B are shown in the following colors: magenta, Sheet-A; cyan, Sheet-B; orange, α-helices H1 and H2. (B) Stereo view of a ribbon model of CcGH131A. Colors are the same as in (A). (C) Structural components unique to CcGH131B. Eight extra components found in CcGH131B (L1-H3, L2, H4, L3, L4, L5-H5, L6, and A10) are categorized into three groups shown in red, blue, and green.
Table 2. Summary of structural similarity search using the DALI server.
| Enzyme | PDB ID | CAZy | Z-score | r.m.s.d. (Å) | Aligned residues | Sequence identity (%) |
| Podospora anserina PaGluc131A | 4LE4 | GH131 | 28.6 | 2.2 | 235 | 27 |
| Coprinopsis cinerea CcGH131A | 3W9A | GH131 | 28.1 | 2.0 | 231 | 30 |
| Hypocrea jecorina endo-β-1,4-glucuronan lyase | 2ZZJ | PL20 | 16.8 | 3.1 | 203 | 10 |
| Bacteroides thetaiotaomicron heparin lyase I | 3IN9 | PL13 | 15.9 | 3.1 | 204 | 13 |
| Sphingomonas sp. A1 alginate lyase | 2ZAA | PL7 | 14.9 | 3.1 | 191 | 9 |
| Bacteroides thetaiotaomicron bimodular hydrolase | 5MQR | GH143 | 14.0 | 3.2 | 181 | 9 |
| Hypocrea jecorina Cip1 | 3ZYP | - | 13.9 | 3.1 | 183 | 9 |
| Clostridium botulinum ganglioside-binding protein | 3N7K | - | 13.6 | 3.4 | 191 | 9 |
| Mycobacterium tuberculosis β-1,3-glucanase | 4WZF | GH16 | 13.3 | 3.6 | 195 | 8 |
Amino acid residue numbers are given at each end of the α-helices and β-strands. β-Strands forming Sheet-A and Sheet-B are labeled A1-A10 and B1-B7, respectively. Five α-helices and six loops are shown as H1-H5 and L1-L6. The eight extra components found in CcGH131B are categorized into three groups shown in red, blue, and green.
When compared to CcGH131A, CcGH131B has eight additional structural components (Fig. 1B). A β-strand A10 is present at the C-terminus, and a long α-helix H4, is located between β-strands A4 and B2. Additionally, there are six long loops, L1-L6, that connect various β-strands in CcGH131B. Short α-helices, H3 and H5, are located in the middle of loops, L1 and L5, respectively, and referred to as L1-H3 and L5-H5 in this paper. These eight additional components are categorized into three groups, shown in red, blue, and green in Fig. 1C and Fig. 2. Loops L2, L4, and L6 (red in Fig. 1C), are located in the rim of the substrate-binding groove and form the first group. Components L3, L5-H5, and H4 (blue in Fig. 1C), form the second group, are found on the opposite side of the groove rim. Due to the presence of the two groups of additional components, the substrate-binding groove in CcGH131B is deeper than that in CcGH131A. The third group consists of β-strand A10 and component L1-H3, which are located on Sheet-A (green in Fig. 1C) and do not interact with the substrate-binding groove.
Cellobiose-bound structure
We first attempted to determine the structure of wild-type CcGH131B in complex with cellobiose and xylooligosaccharide mixture, but only weak electron density was seen in the substrate-binding groove. A Glu residue corresponding to Glu161 in CcGH131B is strictly conserved in the substrate-binding grooves of GH131 proteins and is proposed to function as the catalytic residue [3]. To investigate the substrate preference of CcGH131B, Glu161 was replaced by Ala, and the E161A mutant was soaked in solutions containing glucose, cellobiose, xylose, or xylooligosaccharide mixture. When cellobiose was used, electron density for a cellobiose molecule was clearly seen in the substrate-binding groove (Fig. 3A). The cellobiose molecule formed the same contacts with CcGH131B in both Mol-A and Mol-B in the asymmetric unit (Fig. 3B). Cellobiose potentially binds to subsites −2 and −1, with the two glucose residues described here as Glc−2 and Glc−1. The cellobiose molecule adopted an α-anomeric configuration. A MES buffer molecule was also observed in the same position of the plus subsites of both Mol-A and Mol-B.
(A) Stereo view of the |Fo|−|Fc| omit map, contoured at 3 σ, showing cellobiose and MES (magenta) in the substrate-binding groove. A water molecule (Wat), potentially located at the position of atom Oε1 of Glu161, is also shown. Five residues directly forming hydrogen bonds with cellobiose or MES are illustrated in green. Cyan dotted lines indicate hydrogen bonds. (B) Ribbon model of CcGH131B E161A in an asymmetric unit. Colors: light green, Mol-A; light blue, Mol-B; magenta, cellobiose (Cel); blue, MES; red, polyethylene glycol (PEG).
To analyze the interaction between the protein and ligands, amino acid residues within 4 Å from cellobiose and MES were identified using PyMOL. A total of 17 residues were found in the vicinity of the ligands. The mutated residue Glu161 is also expected to form hydrogen bonds with cellobiose because the position of a water molecule, located 3.0 Å from the atom O5 of Glc−1, corresponds to atom Oε1 of Glu161 in the wild-type protein. In addition, Asp280-Oδ1 forms a hydrogen bond with atom O6 of Glc−2 via a water molecule (Fig. 4).
Symbols: black circle, carbon atom; blue circle, nitrogen atom; red circle, oxygen atom; yellow circle, phosphorus atom; gray circle; water molecule; green dashed line, hydrogen bond; magenta dashed line, salt bridge.
It is intriguing, however, that only four residues, Gln107, Gln156, Glu161, and His167 appear to directly form hydrogen bonds with cellobiose, while Phe110, Phe159, Val281, Val282, Leu264, and Leu266 engage in hydrophobic interactions with cellobiose. Eight water molecules form hydrogen bonds with cellobiose, but five of them do not interact with any amino acid residues of CcGH131B (Fig. 4). In the region of the plus subsites, a phosphate group from the MES molecule forms salt bridges with atoms Nη1 and Nη2 in Arg112, but except for this Arg residue, no other hydrogen bonds are observed between MES and CcGH131B. Leu43, Val45, Val46, Phe98, Asn123, and His260 are involved in hydrophobic interactions with MES.
Comparison to the structure of PaGluc131A
The crystal structure of PaGluc131A in complex with cellotriose (hereafter PaGluc131A-cellotriose) has been reported [3]. To compare the structures of CcGH131B E161A-cellobiose and PaGluc131A-cellotriose, residues listed in Table 3 for the E161A-cellobiose structure and their corresponding residues in PaGluc131A are shown in Fig. 5A and B, respectively. The most striking difference between the two structures is that, in CcGH131B, cellobiose binds to the region of the minus subsites of the substrate-binding groove, while, in PaGluc131A, cellotriose binds to the plus subsites in the groove. Many conserved residues (shown in magenta or orange in Fig. 5A) are present in subsites −1 and −2, but no similarity is found in the plus subsites. Three residues, Trp41, Tyr51, and Trp90, in PaGluc131A appear to engage in stacking interactions with cellotriose, and these residues are conserved in CcGH131A (Table 3). In contrast, no equivalent aromatic residues are found in CcGH131B.
Table 3. Comparison of amino acid residues in the substrate binding site of GH131 enzymes.
| Organism | Coprinopsis cinerea | Coprinopsis cinerea | Podospora anserina |
| Protein name | CcGH131B | CcGH131A | PaGluc131A |
| Protein ID number | CC1G_15039 | CC1G_07166 | PODANS_3_10940 |
| PDB ID | 9K7O | 3W9A | 4LE3 |
| N-terminal CBM | No | CBM1 | CBM1 |
| Subsite −2 | Q156 | Q133 | Q134 |
| G165 | - | - | |
| L264 | L222 | L228 | |
| L266 | I224 | L230 | |
| V281 | - | - | |
| Subsite −1 | V282 | - | - |
| F110 | M94 | M95 | |
| H167 | H140 | H141 | |
| F159 | F136 | F137 | |
| Q107 | N91 | Q93 | |
| Conserved residues | R112 | R96 | R97 |
| E114 | E98 | E99 | |
| E161 | E138 | E139 | |
| H260 | H218 | H224 | |
| Subsite +1 | V46 | I52 | I52 |
| F98 | W90 | W90 | |
| Subsite +2 | L43 | Q49 | Q49 |
| V45 | Y51 | Y51 | |
| N123 | - | - | |
| - | W41 | W41 |
-, No corresponding residue.
Colors: white, cellobiose, MES buffer, and cellotriose; magenta, residues conserved among GH131; orange, residues conserved between CcGH131B and PaGluc131A; green, residues that are unique to CcGH131B or PaGluc131A. The subsite numbers are labeled as −2, −1, +1, +2, and +3. The residue names are indicated for those shown in orange or magenta. In (B), residue names, W41, Y51, and W90, which potentially involve in the substrate binding, are also shown.
Evaluation of activities with saccharides
We evaluated the interaction of CcGH131B with polysaccharides using native affinity PAGE (Fig. 6A-G). The band of CcGH131B without polysaccharide was observed at a position between the 66 kDa and 140 kDa molecular mass markers (Fig. 6A). The molecular mass of CcGH131B is calculated as 35 kDa based on its amino acid sequence, and the discrepancy is probably due to the differences in the isoelectronic points between the molecular weight markers and CcGH131B. The migration positions of CcGH131B in the presence of 1 % Metolose SM-15, 1 % Metolose SM-100, and 1 % carboxymethylcellulose with bands appearing between the 232 kDa and 440 kDa markers (Fig. 6B-D), indicating that CcGH131B binds to cellulosic polysaccharides. The migration positions of CcGH131B did not change in the presence of 1 % xylan, 1 % starch, or 0.5 % laminarin (Fig. 6E-G), even though laminarin has been shown to be hydrolyzed by PaGluc131A [2] and some other GH131 proteins [4].
(A-G) Native affinity PAGE of CcGH131B. Lanes: M, molecular mass marker with numbers indicating molecular sizes in kDa; P, the CcGH131B protein. Electrophoresis was carried out without polysaccharide (A), or in the presence of 1 % methylcellulose Metolose SM-15 (B), 1 % methylcellulose Metolose SM-100 (C), 1 % carboxymethylcellulose (D), 1 % birchwood xylan (E), 1 % starch (F), or 0.5 % laminarin (G). Red arrowheads indicate the migration positions of CcGH131B. (H-N) TLC analysis of polysaccharides after incubation with CcGH131B. Lanes: 1, no CcGH131B added; 2, after incubation with CcGH131B at 37 °C for 20 h. The polysaccharides used are methylcellulose Metolose SM-15 (H), methylcellulose Metolose SM-100 (I), carboxymethylcellulose (J), laminarin (K), starch (L), birchwood xylan (M), or Avicel PH101 (N).
We tested whether CcGH131B could degrade the polysaccharides using TLC (Fig. 6H-N). For Metolose SM-15, Metolose SM-100, carboxymethylcellulose, laminarin, starch, and xylan, spots were observed at the origin before incubation (Lane 1, Fig. 6H-M). After incubation, these spots remained unchanged (Lane 2, Fig. 6H-M), indicating no detectable activity of CcGH131B, as no other spots appeared at different positions on TLC. The insoluble polysaccharide Avicel PH101 showed no spot at the origin before the incubation (Fig. 6), as the supernatant was spotted on TLC (Lane 1, Fig. 6N). No significant spot was observed on TLC after the incubation (Lane 2, Fig. 6N), suggesting that CcGH131B did not release soluble saccharides from Avicel PH101. To test whether CcGH131B could act on p-nitrophenyl β-D-cellobioside, changes in the absorbance were monitored as described in Materials and Methods. The absorbance after the incubation for 9 h was almost 0, showing that no activity was detected for p-nitrophenyl β-D-cellobioside.
In this study, CC1G_15039, a protein belonging to GH131, was designated CcGH131B, and the structure and some properties were investigated. The structure of E161A-cellobiose showed that cellobiose bound to the minus subsites while the MES buffer molecule was observed in the plus subsites. The amino acid residues forming the plus subsites exhibited low homology to those of PaGluc131A, and hydrophobic residues such as Leu43, Val45, Val46, and Phe98 were present in the vicinity of the MES molecule. This observation suggests that the region of the minus subsites binds to cellulosic molecules, while that of the plus subsites likely interacts with hydrophobic molecules and the affinity for carbohydrates appears to be weak.
The results of native affinity PAGE showed that CcGH131B interacted with cellulosic polysaccharides, methylcellulose and carboxymethylcellulose. To probe the enzymatic activity, CcGH131B was incubated with methylcellulose and carboxymethylcellulose, but no degradation of the polysaccharides was detected by TLC analysis. One reason for this might be that the reaction conditions were not optimal. The enzymatic activities of seven GH131 proteins have been investigated, and all were found to be active on substrates with β-1,4-, β-1,3-, and mixed β-1,3-/-1,4-glucosidic linkages, though their individual activities varied [4]. PaGluc131A has been reported to display exo-β-1,3-/-1,6- and endo-β-1,4-activities toward β-glucans [2], and the activities (sum of released saccharides in nmol per nmol of PaGluc131A with 1 % polysaccharide for 24 h) for carboxymethylcellulose (β-1,4-glucan), curdlan (β-1,3-glucan), and laminarin (β-1,3-/-1,6-glucan) are 126, 271, and 434, respectively [4]. In contrast, some endoglucanases have been determined to release about 3-100 µmol of reducing end per mg (approximately 10-30 nmol) of enzyme for 1 min from carboxymethylcellulose [27, 28, 29], and these activities are much higher than those of GH131 proteins.
A phylogenetic analysis of GH131 indicated that GH131 proteins are classified into four groups, Clades A-D [4]. Proteins from oomycetes are grouped into Clade A, while Clades B and C contain proteins from both basidiomycetes and ascomycetes, and Clade D contains proteins from basidiomycetes. CcGH131A and CcGH131B fall into Clades B and D, respectively. A GH131 protein, PsGluc131B, from Pycnoporus sanguineus, has been classified into Clade D, and enzymatic analysis shows that PsGluc131B prefers mixed linked β-1,3-/-1,4-glucan over β-1,4-glucan and β-1,3-glucan. We aligned the amino acid sequences of CcGH131B, CcGH131A, and several GH131 proteins described in the literature [4], which have been classified into Clades B, D, and C (Fig. S1; see J. Appl. Glycosci. Web site). In CcGH131B, the loops L1-L6, α-helices H4 and H5, and β-strand A10 were structurally characteristic based on the comparison with CcGH131A. These components were largely conserved in the Clade D protein PsGluc131B, while parts of these components, such as L2, L3, L4, and L5-H5, were found in the Clade C proteins. In contrast, in the Clade B proteins, these loops, α-helices, and β-strand were shorter or absent. Among the five amino acid residues in the plus subsites of CcGH131B (Table 3), Leu43, Val45, and Val46 are present in the Clade D protein PsGluc131B, and Phe98 is conserved in the Clades D and C proteins, while Asn123 is unique to CcGH131B. These results suggest that the key amino acid residues of CcGH131B are distinct from those of the Clades B and C proteins and even from those of the Clade D protein PsGluc131B.
GH131 proteins act on various β-glucan polysaccharides, but the primary role in the degradation of cellulosic biomass is still not fully understood. The structural features of CcGH131B indicate that the plus subsites of CcGH131B are markedly different from those of CcGH131A and PaGluc131A, suggesting that the primary substrate of CcGH131B is likely different from those of CcGH131A and PaGluc131A. The DALI search indicated that CcGH131B shares distant homology with PL20 endo-β-1,4-glucuronan lyase and T. reesei Cip1. It has been reported that Cip1 promotes the enzymatic hydrolysis of pretreated lignocellulose [30], raising the possibility that CcGH131B may interact with such macromolecules.
There are two possible reasons why no enzyme activity of CcGH131B for β-glucans was observed in this study. First, the reaction conditions used might not have been optimal for CcGH131B, as described above. Another possible reason is that the primary function of CcGH131B may not be as a β-glucan hydrolase. The substrate-binding groove of CcGH131B is strikingly different from that of PaGluc131A due to the presence of extra loops and α-helices. Additionally, the plus subsites of CcGH131B do not appear to be suitable for carbohydrate binding, as many hydrophobic amino acid residues are located in these sites.
This study presents insights into the structural and functional properties of CcGH131B. The crystal structure revealed that CcGH131B possesses a β-jelly roll fold similar to other GH131 proteins but also features unique structural elements, such as additional loops and α-helices, contributing to a deeper substrate-binding groove compared to CcGH131A and PaGluc131A. The structure of E161A-cellobiose showed that the region of the minus subsites is likely to bind to the cellulosic molecules, while that of the plus subsites appeared to interact with hydrophobic molecules rather than with carbohydrates. Despite binding to cellulosic polysaccharides, such as methylcellulose and carboxymethylcellulose, CcGH131B displayed no detectable enzymatic activity under the tested conditions. These results suggest that the substrate specificity of CcGH131B is likely to be different from those of CcGH131A and PaGluc131A. Future studies exploring a broader range of substrates and conditions will be essential to fully elucidate the enzymatic function and physiological role of CcGH131B in biomass degradation.
The authors declare no conflict of interest.
We thank Masahiro Hayashi, Shunsaku Okuyama, Ryoichi Ishikawa, and Hiyori Uchida for the crystallization and determination of the structures. We also thank Shin-Etsu Chemical Co. Ltd. for providing Metolose SM-15 and Metolose SM-100. This work was supported in part by a Grant-in-Aid for Scientific Research (24K08698 to T.T., 24K17938 to Y.K., 23K18046 to M.Y.) from the Japan Society for the Promotion of Science and SPRING (JPMJSP2116 to Y.S.) from Japan Science and Technology Agency (JST). This work has been performed under the approval of the Photon Factory Program Advisory Committee (2023G505).
